Abstract
Cold storage is a commonly adopted technology to maintain quality and extend shelf life; however, depending on the cultivar, ethylene production can be enhanced by short-term cold treatments. In this study, index of absorbance difference (IAD) values, which reflect skin chlorophyll concentrations of cold-influenced ripening patterns, of ‘Gala’ apples were investigated. Fruit that were untreated or treated with aminoethoxyvinylglycine (AVG), which is a plant growth regulator that inhibits ethylene production of fruit, were harvested from a commercial orchard. Two IAD value categories 0.6 to 0.8 and 0.2 to 0.4 were chosen from a single harvest in 2019, whereas in 2020, the fruit in categories 0.6 to 0.8 and 0.2 to 0.4 were chosen for two harvests separated by 1 week, respectively. In 2019, only ethylene production was measured. In 2020, ethylene production, respiration, internal ethylene concentration (IEC), flesh firmness, 1-aminocyclopropane-1-carboxylate (ACC), and malonyl 1-aminocyclopropane-1-carboxylate (MACC) concentrations of fruit kept in air at 20 °C or stored at 0.5 °C for 21 days and then transferred to 20 °C were measured. The results were different from those described in the literature for cold-enhanced ethylene production of ‘Gala’ apples. Although ethylene production occurred without cold exposure of untreated fruit, exposure of fruit to 0.5 °C for 21 days resulted in more rapid and higher ethylene production rates and IEC than for fruit kept at only 20 °C. Ethylene production was suppressed by the preharvest AVG treatment, especially in 2020. The rates of respiration and softening of non-AVG treated fruit were enhanced by cold treatment. The effects of cold treatment were more significant for less mature fruit as indicated by higher IAD values.
‘Gala’ is a major apple cultivar grown worldwide that is mostly planted as red sports, such as Royal, Brookfield, Fulford, and Galaxy. The red sports tend to have similar maturation profiles (Argenta et al. 2021; Walsh and Volz 1990), but they allow earlier harvests and strip picking, which is not recommended for the original ‘Gala’ or lower-colored sports (Brookfield et al. 1993). Although Gala fruit are harvested early relative to typical long-term storage cultivars such as Red Delicious, Granny Smith, and Fuji, they are often stored long-term under controlled atmosphere conditions.
The plant growth regulator AVG, as the commercial product ReTain®, is commonly used to manage apple harvest in many growing regions (Doerflinger et al. 2019; Petri et al. 2007; Phan-Thien et al. 2004). The effect of AVG on apple fruit is generally assumed to act via inhibition of ACC synthase, which catalyzes the conversion of methionine to ACC, the precursor to ethylene in the ethylene biosynthetic pathway (Boller et al. 1979). AVG binds to pyridoxal-5′-phosphate; therefore, action through nonethylene pathways cannot be excluded (Le Deunff et al. 2019). Regardless, inhibited or delayed ethylene production in AVG-treated fruit is associated with decreased losses caused by preharvest drop and delayed maturation and, subsequently, delayed harvest that allows the fruit size to increase (Arseneault and Cline 2016; Doerflinger et al. 2019; Phan-Thien et al. 2004; Schupp and Greene 2004; Yuan and Li 2008). AVG can also reduce the incidence of disorders such as stem end flesh browning of ‘Gala’ apples during long-term storage (Nock et al. 2019; Watkins and Mattheis 2019).
Cold storage is the major technology that is used to maintain the quality of apple fruit. However, the effects of cold storage on ethylene production of the fruit as well as on its production when fruit are transferred to warmer temperatures after cold storage can be complex. The responses of apple fruit to temperature are greatly influenced by cultivar and can be analogous to that of winter pear fruit (Lelievre et al. 1997; Villalobos-Acuna and Mitcham 2008), which typically need exposure to chilling temperatures to induce normal ripening. Of five apple cultivars investigated, ethylene production of Golden Delicious apples was more rapid and uniform in fruit stored at 3 °C than at 18 to 20 °C, and production was higher at 3 °C than at 18 °C (Knee et al. 1983). Larrigaudiere et al. (1997) found that ethylene production of ‘Royal Gala’, ‘Starking Delicious’, and ‘Granny Smith’ apples exposed to 1 °C for 10 d and transferred to 20 °C was delayed, depressed, or stimulated, respectively. Recovery of ethylene production was faster for ‘Starking Delicious’ and ‘Granny Smith’ than for ‘Royal Gala’. Of the three cultivars, only Royal Gala produced ethylene at low temperature and did not require prolonged cold storage. Exposure to 0 °C induced ethylene production of ‘Braeburn’, ‘Fuji’, ‘Granny Smith’, and ‘Lady Williams’ apples; the longer the exposure, the greater the stimulation (Jobling and McGlasson 1995; Jobling et al. 1991, 2003; Tian et al. 2002). ‘Braeburn’ apples could recover from 1-methylcyclopropene inhibition of ethylene perception at 0 °C, but not 20 °C (Tian et al. 2002). Cold storage temperature induction of ethylene production in apple fruit was associated with increased ACC and MACC concentrations (Jobling et al. 1991; Knee et al. 1983; Lara and Vendrell 2003; Vilaplana et al. 2007), increased activity of 1-aminocyclopropane-1-carboxylate synthase and ACC oxidase, and associated gene expression (Lara and Vendrell 2000, 2003; Larrigaudiere and Vendrell 1993; Tian et al. 2002).
Little is known about the effects of plant growth regulators and fruit maturity on the responses of apples to exposure to cold storage. During these experiments, we used AVG treatment of ‘Gala’ in the field and used the difference of absorbance meter to separate fruit maturity groups. The difference of absorbance meter determines the absorbance difference between 670 nm and 720 nm wavelengths) (Ziosi et al. 2008) and provides an absorbance difference index (IAD) value as an index measure of chlorophyll a; high values indicate higher chlorophyll concentrations. The difference of absorbance meter has been used widely as an additional tool to determine harvest because it allows measurement of chlorophyll loss in the fruit skin during the maturation phase (Cocetta et al. 2017; DeLong et al. 2020; Doerflinger et al. 2014; Nyasordzi et al. 2013; Peifer et al. 2018), including that of ‘Gala’ (Sadar and Zanella 2019; Toivonen and Hampson 2014; Toivonen and Lannard 2021; Toivonen et al. 2014). These studies have focused on bulk samples of fruit and development of correlations with other harvest indices such as ethylene production, starch pattern index, and flesh firmness. Farneti et al. (2015) used IAD values to tailor 1-methylcyclopropene treatment based on the risk of superficial scald development for ‘Granny Smith’ and ‘Cripps Pink’ apples. However, few studies have used IAD values to investigate the maturation stage of fruit as a way to explore physiological processes in apple fruit.
The objectives of the current study were to assess the effects of a 21-d cold storage treatment on ethylene production of ‘Gala’ using fruit from trees treated with AVG separated into two maturity classes at harvest based on IAD values.
Materials and Methods
Preharvest PGR treatment.
High density ‘Gala’ apple trees (tall spindle) on a commercial orchard in Western New York with 2200 trees per hectare were used for this 2-year experiment. Four sets of two (2019) or four trees (2020) per replicate were arranged in a randomized complete block design, with buffer zones of three trees between treatment plots. AVG (ReTain®; Valent BioScience Corporation, Libertyville, IL, USA) at 0.25 g⋅L−1 was sprayed at 410 g per hectare 4 weeks before the first anticipated commercial harvest each year. AVG was applied in the early morning using a CO2 pressurized backpack sprayer (Bellspray, Opelousas, LA, USA) fitted with a TeeJet 8004VS flat fan nozzle (Spraying Systems, Wheaton, IL, USA). Untreated trees were not sprayed.
Harvest and categorization by IAD values.
All fruit from each two-tree replicate of the untreated and AVG-treated trees were harvested on 18 Sep 2019, 8 Sep 2020 [harvest 1 (H1)], and 21 Sep 2020 [harvest 2 (H2)]. The fruit were transported to the Cornell postharvest laboratory on the day of harvest. Then, fruit were sorted by IAD value using a handheld delta absorbance meter (Sinteleia, Bologna, Italy). The IAD values represented the average of the blushed and an unblushed sides of each apple.
Fruit were separated into categories of 0 to 0.2, 0.21 to 0.4, 0.41 to 0.6, 0.61 to 0.8, and 0.81 to 1. Fruit from each treatment was combined. The categories chosen for research were based on fruit numbers available for untreated and AVG-treated fruit and differences of maturity. The categories of 0.2 to 0.4 and 0.6 to 0.8 were chosen in both years. However, in 2019, both categories were used in a single harvest, whereas in 2020, the fruit in categories 0.6 to 0.8 and 0.2 to 0.4 were chosen for H1 and H2, respectively. Fruit numbers per category were 10 in 2019 and 240 at each harvest in 2020.
Then, the fruit of each category for both untreated and AVG treatments were divided into two sets. One set was kept at 20 °C, whereas the other was stored at 0.5 °C and 85% relative humidity for 21 d before being transferred to 20 °C. Fruit with or without cold storage were used for assessments of ethylene production, respiration rate, internal ethylene concentration (IEC), flesh firmness, and ACC and MACC concentrations.
Ethylene production and respiration rates.
Ethylene production and respiration rate were measured using weighed individual fruit (5 per treatment in 2019 and 6 per treatment in 2020) on a flow board system. Each fruit was kept in a 1-quart polyethylene container (Cambro, Huntington Beach, CA, USA) with an air flow rate of 0.6 L⋅h−1. The air flow rate was measured using a ADM1000 gas flowmeter (J&W Scientific, Folsom, CA, USA). Fruit in 2019 were only used for ethylene production measurement for 18 d, without further assessments; in 2020, fruit were used for measurements of ethylene and CO2 production rates for up to 21 d. Measurements were usually daily.
The 1-mL gas samples from the containers were used for measurements of ethylene and CO2 and subsequent calculations of production rates of each fruit. Containers without fruit were used as blanks. Ethylene was measured using Hewlett-Packard 5890 series II gas chromatography (Hewlett-Packard, Wilmington, DE, USA) with a flame ionization detector and stainless-steel column packed with 60/80 mesh Alumina F-1 (2 m × 2 mm inner diameter). CO2 was measured using a CA-10 carbon dioxide analyzer (Sable Systems International, Las Vegas, NV, USA). Ethylene and CO2 production were calculated as (sample − blank) × R/W, where R = the air flow rate (L⋅h−1) and W = the fruit weight (kg).
IEC, flesh firmness, and ACC and MACC concentrations.
Six fruit considered independent replicates were sampled at 2- to 3-d intervals. The IEC was measured using 1-mL core samples with gas chromatography as described previously. The results are presented as μL⋅L−1. Flesh firmness was measured opposite of the equatorial region of each using a fruit texture analyzer fitted with a 11.1-mm diameter stainless probe (Guss Manufacturing Pty. Ltd., Strand, South Africa). Firmness was recorded in Newtons (N). For quantification of ACC and MACC, peel samples were obtained from 0 to 21 days, frozen in liquid nitrogen, and stored at −80 °C. The tissues were ground into fine powder with an IKA® A11 basic analytical mill (IKA® Works, Inc., Wilmington, NC, USA) under liquid nitrogen. The powdered samples were stored at −80 °C. Extraction and assay of ACC and MACC were performed using 1-g samples according to the method of Bulens et al. (2011).
Statistical analysis.
Results are shown as means ± SE of independent determinations. The significance of differences between means were determined by Fisher’s least significant difference values (P = 0.05). Changes of flesh firmness over time were analyzed by linear regression. Statistical analyses were performed with Office 365 Excel and JMP® Pro 15 (SAS Institute Inc., Cary, NC, USA).
Results
Ethylene production and respiration rate.
During year 1, only fruit that were either untreated or AVG-treated were studied. Fruit were stored at 20 °C without cold storage. Categories of fruit maturity based on IAD values of 0.6 to 0.8 (Fig. 1A) and 0.2 to 0.4 (Fig. 1B), indicating higher and lower chlorophyll contents, respectively, were used to compare the ethylene production of fruit from the two treatments.
Ethylene production (μL⋅kg−1⋅h−1) of untreated control and aminoethoxyvinylglycine (AVG)-treated ‘Gala’ apples with absorbance difference index (IAD) values of 0.6 to 0.8 (A) and 0.2 to 0.4 (B) kept at 20 °C after harvest in 2019. ( 
) Control fruit. ( 
) Fruit treated with AVG. Bars represent ± SE (n = 5), whereas the least significant difference values represent P < 0.05. P values: field treatment <0.0001; IAD values = 0.0003; field treatment × IAD values = 0.1261.
Citation: HortScience 58, 5; 10.21273/HORTSCI17074-23
Ethylene production (μL⋅kg−1⋅h−1) of untreated control and aminoethoxyvinylglycine (AVG)-treated ‘Gala’ apples with absorbance difference index (IAD) values of 0.6 to 0.8 (A) and 0.2 to 0.4 (B) kept at 20 °C after harvest in 2019. ( ) Control fruit. ( ) Fruit treated with AVG. Bars represent ± SE (n = 5), whereas the least significant difference values represent P < 0.05. P values: field treatment <0.0001; IAD values = 0.0003; field treatment × IAD values = 0.1261.
Citation: HortScience 58, 5; 10.21273/HORTSCI17074-23
Ethylene production (μL⋅kg−1⋅h−1) of untreated control and aminoethoxyvinylglycine (AVG)-treated ‘Gala’ apples with absorbance difference index (IAD) values of 0.6 to 0.8 (A) and 0.2 to 0.4 (B) kept at 20 °C after harvest in 2019. ( ) Control fruit. ( ) Fruit treated with AVG. Bars represent ± SE (n = 5), whereas the least significant difference values represent P < 0.05. P values: field treatment <0.0001; IAD values = 0.0003; field treatment × IAD values = 0.1261.
Citation: HortScience 58, 5; 10.21273/HORTSCI17074-23
Treatment with AVG delayed the increase of ethylene production by ∼2 d and 4 d in the 0.6 to 0.8 and 0.2 to 0.4 categories, respectively. Ethylene production of the untreated fruit was higher in the 0.6 to 0.8 category than in the 0.2 to 0.4 category, with averages of 40.80 and 27.78 μL⋅kg−1⋅h−1, respectively, over days 12 to 18. The average ethylene production values for AVG-treated fruit were 6.31 μL⋅kg−1⋅h−1 and 3.29 μL⋅kg−1⋅h−1 for the 0.6 to 0.8 and 0.2 to 0.4 categories, respectively, over the same time period at 20 °C.
The same maturity categories were selected in year 2 to investigate the effect of cold storage treatment on the ethylene production (Fig. 2) and respiration rate (Fig. 3) of untreated and AVG-treated fruit. However, the 0.6 to 0.8 and 0.2 to 0.4 categories differed by harvest (H1 and H2, respectively). For untreated fruit that were not exposed to cold treatment, ethylene production of fruit in the 0.6 to 0.8 category started to increase on day 7, whereas an increase in ethylene production was found for the 0.2 to 0.4 category fruit after day 13. Overall, ethylene production of fruit in the 0.6 to 0.8 category was higher than that in the 0.2 to 0.4 category (Fig. 2A, B; Table 1). Cold storage of untreated fruit resulted in a rapid increase of ethylene production. Overall, ethylene production was higher in fruit of the 0.6 to 0.8 category than in that of the 0.2 to 0.4 category, with an average of 117% higher from days 5 to 21. AVG suppressed ethylene production of the fruit over 21 d and was unaffected by cold storage (Fig. 2A, B).
Ethylene production (μL⋅kg−1⋅h−1) of untreated control and aminoethoxyvinylglycine (AVG)-treated ‘Gala’ apples with absorbance difference index (IAD) values for harvest 1 (H1) of 0.6 to 0.8 (A) and for harvest 2 (H2) of 0.2 to 0.4 (B) kept at 20 °C after harvest or after storage at 0.5 °C for 3 weeks in 2020. The time of fruit harvest or transfer from 0.5 °C to 20 °C is represented as day 0. () Control fruit. () Fruit treated with AVG. (
) Control fruit (0.5–20 °C). (
) Fruit treated with AVG (0.5–20 °C). Bars represent ± SE (n = 6), whereas least significant difference values represent P < 0.05.
Citation: HortScience 58, 5; 10.21273/HORTSCI17074-23
Ethylene production (μL⋅kg−1⋅h−1) of untreated control and aminoethoxyvinylglycine (AVG)-treated ‘Gala’ apples with absorbance difference index (IAD) values for harvest 1 (H1) of 0.6 to 0.8 (A) and for harvest 2 (H2) of 0.2 to 0.4 (B) kept at 20 °C after harvest or after storage at 0.5 °C for 3 weeks in 2020. The time of fruit harvest or transfer from 0.5 °C to 20 °C is represented as day 0. () Control fruit. () Fruit treated with AVG. () Control fruit (0.5–20 °C). () Fruit treated with AVG (0.5–20 °C). Bars represent ± SE (n = 6), whereas least significant difference values represent P < 0.05.
Citation: HortScience 58, 5; 10.21273/HORTSCI17074-23
Ethylene production (μL⋅kg−1⋅h−1) of untreated control and aminoethoxyvinylglycine (AVG)-treated ‘Gala’ apples with absorbance difference index (IAD) values for harvest 1 (H1) of 0.6 to 0.8 (A) and for harvest 2 (H2) of 0.2 to 0.4 (B) kept at 20 °C after harvest or after storage at 0.5 °C for 3 weeks in 2020. The time of fruit harvest or transfer from 0.5 °C to 20 °C is represented as day 0. () Control fruit. () Fruit treated with AVG. () Control fruit (0.5–20 °C). () Fruit treated with AVG (0.5–20 °C). Bars represent ± SE (n = 6), whereas least significant difference values represent P < 0.05.
Citation: HortScience 58, 5; 10.21273/HORTSCI17074-23
Respiration rate (mg CO2 kg−1⋅h−1) of untreated control and aminoethoxyvinylglycine (AVG)-treated ‘Gala’ apples with absorbance difference index (IAD) values for harvest 1 (H1) of 0.6 to 0.8 (A) and for harvest 2 (H2) of 0.2 to 0.4 (B) kept at 20 °C after harvest or after storage at 0.5 °C for 3 weeks in 2020. The time of fruit harvest or transfer from 0.5 °C to 20 °C is represented as day 0. () Control fruit. () Fruit treated with AVG. () Control fruit (0.5–20 °C). () Fruit treated with AVG (0.5–20 °C). Bars represent ± SE (n = 6), whereas least significant difference values represent P < 0.05.
Citation: HortScience 58, 5; 10.21273/HORTSCI17074-23
Respiration rate (mg CO2 kg−1⋅h−1) of untreated control and aminoethoxyvinylglycine (AVG)-treated ‘Gala’ apples with absorbance difference index (IAD) values for harvest 1 (H1) of 0.6 to 0.8 (A) and for harvest 2 (H2) of 0.2 to 0.4 (B) kept at 20 °C after harvest or after storage at 0.5 °C for 3 weeks in 2020. The time of fruit harvest or transfer from 0.5 °C to 20 °C is represented as day 0. () Control fruit. () Fruit treated with AVG. () Control fruit (0.5–20 °C). () Fruit treated with AVG (0.5–20 °C). Bars represent ± SE (n = 6), whereas least significant difference values represent P < 0.05.
Citation: HortScience 58, 5; 10.21273/HORTSCI17074-23
Respiration rate (mg CO2 kg−1⋅h−1) of untreated control and aminoethoxyvinylglycine (AVG)-treated ‘Gala’ apples with absorbance difference index (IAD) values for harvest 1 (H1) of 0.6 to 0.8 (A) and for harvest 2 (H2) of 0.2 to 0.4 (B) kept at 20 °C after harvest or after storage at 0.5 °C for 3 weeks in 2020. The time of fruit harvest or transfer from 0.5 °C to 20 °C is represented as day 0. () Control fruit. () Fruit treated with AVG. () Control fruit (0.5–20 °C). () Fruit treated with AVG (0.5–20 °C). Bars represent ± SE (n = 6), whereas least significant difference values represent P < 0.05.
Citation: HortScience 58, 5; 10.21273/HORTSCI17074-23
P values from the analysis of variance of the effects of preharvest treatment, absorbance difference index (IAD) category, storage temperature, and their interactions on ethylene production, internal ethylene concentration (IEC), respiration rate, flesh firmness, and 1-aminocyclopropane-1-carboxylate (ACC), and malonyl ACC (MACC) concentrations of ‘Gala’ fruit.
Overall, the respiration rate of untreated fruit in the 0.6 to 0.8 category had an average of 4.67 mg CO2 kg−1⋅h−1 compared with 6.71 mg CO2 kg−1⋅h−1 in the 0.2 to 0.4 category. The rates remained low before increasing after 16 d in the 0.6 to 0.8 category, whereas they increased within a few days in the 0.2 to 0.4 category (Fig. 3A, B). The respiration rates of untreated fruit were usually higher than the respiration rates of the AVG-treated fruit (average of 1.59 mg CO2 kg−1⋅h−1 in the 0.6 to 0.8 category; average of 4.51 mg CO2 kg−1⋅h−1 in the 0.2 to 0.4 category), although less consistently in the 0.2 to 0.4 category than in the 0.6 to 0.8 category (Fig. 3A, B; Table 1).
Respiration rates were stimulated by cold storage, although patterns were affected by the IAD category (Fig. 3A, B). For cold-stored fruit in the 0.6 to 0.8 category, the untreated control fruit had much higher respiration rates than AVG-treated fruit (Fig. 3A) (14.55 mg CO2 kg−1⋅h−1 and 5.46 mg CO2 kg−1⋅h−1, respectively). For cold-stored fruit in the 0.2 to 0.4 category, initial respiration rates of the control and AVG-treated fruit were both higher initially, but rates of the AVG-treated fruit declined more rapidly compared with those of the control fruit.
IEC, flesh firmness, ACC and MACC concentrations.
Parallel to the ethylene production and respiration rate measurements in year 2, fruit from the maturity categories 0.6 to 0.8 and 0.2 to 0.4 were used to investigate the effects of AVG and cold storage on IEC (Fig. 4), flesh firmness (Fig. 5), as well as ACC and MACC concentrations in the skin (Fig. 6). The analysis of variance-derived P value for each factor is shown in Table 1.
Internal ethylene concentration (IEC) (μL⋅L−1) of untreated control and aminoethoxyvinylglycine (AVG)-treated ‘Gala’ apples with absorbance difference index (IAD) values for harvest 1 (H1) of 0.6 to 0.8 (A) and for harvest 2 (H2) of 0.2 to 0.4 (B) kept at 20 °C after harvest or after storage at 0.5 °C for 3 weeks in 2020. The time of fruit harvest or transfer from 0.5 °C to 20 °C is represented as day 0. () Control fruit. () Fruit treated with AVG. () Control fruit (0.5–20 °C). () Fruit treated with AVG (0.5–20 °C). Bars represent ± SE (n = 6), whereas least significant difference values represent P < 0.05.
Citation: HortScience 58, 5; 10.21273/HORTSCI17074-23
Internal ethylene concentration (IEC) (μL⋅L−1) of untreated control and aminoethoxyvinylglycine (AVG)-treated ‘Gala’ apples with absorbance difference index (IAD) values for harvest 1 (H1) of 0.6 to 0.8 (A) and for harvest 2 (H2) of 0.2 to 0.4 (B) kept at 20 °C after harvest or after storage at 0.5 °C for 3 weeks in 2020. The time of fruit harvest or transfer from 0.5 °C to 20 °C is represented as day 0. () Control fruit. () Fruit treated with AVG. () Control fruit (0.5–20 °C). () Fruit treated with AVG (0.5–20 °C). Bars represent ± SE (n = 6), whereas least significant difference values represent P < 0.05.
Citation: HortScience 58, 5; 10.21273/HORTSCI17074-23
Internal ethylene concentration (IEC) (μL⋅L−1) of untreated control and aminoethoxyvinylglycine (AVG)-treated ‘Gala’ apples with absorbance difference index (IAD) values for harvest 1 (H1) of 0.6 to 0.8 (A) and for harvest 2 (H2) of 0.2 to 0.4 (B) kept at 20 °C after harvest or after storage at 0.5 °C for 3 weeks in 2020. The time of fruit harvest or transfer from 0.5 °C to 20 °C is represented as day 0. () Control fruit. () Fruit treated with AVG. () Control fruit (0.5–20 °C). () Fruit treated with AVG (0.5–20 °C). Bars represent ± SE (n = 6), whereas least significant difference values represent P < 0.05.
Citation: HortScience 58, 5; 10.21273/HORTSCI17074-23
Flesh firmness (N) of untreated control and aminoethoxyvinylglycine (AVG)-treated ‘Gala’ apples with absorbance difference index (IAD) values for harvest 1 (H1) of 0.6 to 0.8 (A) and for harvest 2 (H2) of 0.2 to 0.4 (B) kept at 20 °C after harvest or after storage at 0.5 °C for 3 weeks in 2020. The time of fruit harvest or transfer from 0.5 °C to 20 °C is represented as day 0. () Control fruit. () Fruit treated with AVG. () Control fruit (0.5–20 °C). () Fruit treated with AVG (0.5–20 °C). Bars represent ± SE (n = 6), whereas least significant difference values represent P < 0.05. In the 0.6 to 0.8 category, linear regressions between fruit firmness and storage time at 20 °C are as follows: control y = −0.8271x + 82.247, R2 = 0.686; AVG y = −0.3361x + 81.229, R2 = 0.3546; control (0.5–20 °C) y = −0.7815x + 70.85, R2 = 0.7761; and AVG (0.5–20 °C) y = −0.2015x + 75.905, R2 = 0.4165. In the 0.2 to 0.4 category, linear regressions between fruit firmness and storage time at 20 °C are as follows: control y = −0.8295x + 74.503, R2 = 0.7179; AVG: y = −0.3874x + 77.094, R2 = 0.471; control (0.5–20 °C): y = −1.0151x + 69.264, R2 = 0.9096; and AVG (0.5–20 °C): y = −0.4403x + 69.831, R2 = 0.5407.
Citation: HortScience 58, 5; 10.21273/HORTSCI17074-23
Flesh firmness (N) of untreated control and aminoethoxyvinylglycine (AVG)-treated ‘Gala’ apples with absorbance difference index (IAD) values for harvest 1 (H1) of 0.6 to 0.8 (A) and for harvest 2 (H2) of 0.2 to 0.4 (B) kept at 20 °C after harvest or after storage at 0.5 °C for 3 weeks in 2020. The time of fruit harvest or transfer from 0.5 °C to 20 °C is represented as day 0. () Control fruit. () Fruit treated with AVG. () Control fruit (0.5–20 °C). () Fruit treated with AVG (0.5–20 °C). Bars represent ± SE (n = 6), whereas least significant difference values represent P < 0.05. In the 0.6 to 0.8 category, linear regressions between fruit firmness and storage time at 20 °C are as follows: control y = −0.8271x + 82.247, R2 = 0.686; AVG y = −0.3361x + 81.229, R2 = 0.3546; control (0.5–20 °C) y = −0.7815x + 70.85, R2 = 0.7761; and AVG (0.5–20 °C) y = −0.2015x + 75.905, R2 = 0.4165. In the 0.2 to 0.4 category, linear regressions between fruit firmness and storage time at 20 °C are as follows: control y = −0.8295x + 74.503, R2 = 0.7179; AVG: y = −0.3874x + 77.094, R2 = 0.471; control (0.5–20 °C): y = −1.0151x + 69.264, R2 = 0.9096; and AVG (0.5–20 °C): y = −0.4403x + 69.831, R2 = 0.5407.
Citation: HortScience 58, 5; 10.21273/HORTSCI17074-23
Flesh firmness (N) of untreated control and aminoethoxyvinylglycine (AVG)-treated ‘Gala’ apples with absorbance difference index (IAD) values for harvest 1 (H1) of 0.6 to 0.8 (A) and for harvest 2 (H2) of 0.2 to 0.4 (B) kept at 20 °C after harvest or after storage at 0.5 °C for 3 weeks in 2020. The time of fruit harvest or transfer from 0.5 °C to 20 °C is represented as day 0. () Control fruit. () Fruit treated with AVG. () Control fruit (0.5–20 °C). () Fruit treated with AVG (0.5–20 °C). Bars represent ± SE (n = 6), whereas least significant difference values represent P < 0.05. In the 0.6 to 0.8 category, linear regressions between fruit firmness and storage time at 20 °C are as follows: control y = −0.8271x + 82.247, R2 = 0.686; AVG y = −0.3361x + 81.229, R2 = 0.3546; control (0.5–20 °C) y = −0.7815x + 70.85, R2 = 0.7761; and AVG (0.5–20 °C) y = −0.2015x + 75.905, R2 = 0.4165. In the 0.2 to 0.4 category, linear regressions between fruit firmness and storage time at 20 °C are as follows: control y = −0.8295x + 74.503, R2 = 0.7179; AVG: y = −0.3874x + 77.094, R2 = 0.471; control (0.5–20 °C): y = −1.0151x + 69.264, R2 = 0.9096; and AVG (0.5–20 °C): y = −0.4403x + 69.831, R2 = 0.5407.
Citation: HortScience 58, 5; 10.21273/HORTSCI17074-23
The 1-aminocyclopropane-1-carboxylate (ACC) (nmol⋅g−1) (A and B) and malonyl ACC (MACC) (nmol⋅g−1) (C and D) of untreated control and aminoethoxyvinylglycine (AVG)-treated ‘Gala’ apples with absorbance difference index (IAD) values for harvest 1 (H1) of 0.6 to 0.8 (A) and for harvest 2 (H2) of 0.2 to 0.4 (B) kept at 20 °C after harvest or after storage at 0.5 °C for 3 weeks in 2020. The time of fruit harvest or transfer from 0.5 °C to 20 °C is represented as day 0. () Control fruit. () Fruit treated with AVG. () Control fruit (0.5–20 °C). () Fruit treated with AVG (0.5–20 °C). Bars represent ± SE (n = 6), whereas least significant difference values represent P < 0.05.
Citation: HortScience 58, 5; 10.21273/HORTSCI17074-23
The 1-aminocyclopropane-1-carboxylate (ACC) (nmol⋅g−1) (A and B) and malonyl ACC (MACC) (nmol⋅g−1) (C and D) of untreated control and aminoethoxyvinylglycine (AVG)-treated ‘Gala’ apples with absorbance difference index (IAD) values for harvest 1 (H1) of 0.6 to 0.8 (A) and for harvest 2 (H2) of 0.2 to 0.4 (B) kept at 20 °C after harvest or after storage at 0.5 °C for 3 weeks in 2020. The time of fruit harvest or transfer from 0.5 °C to 20 °C is represented as day 0. () Control fruit. () Fruit treated with AVG. () Control fruit (0.5–20 °C). () Fruit treated with AVG (0.5–20 °C). Bars represent ± SE (n = 6), whereas least significant difference values represent P < 0.05.
Citation: HortScience 58, 5; 10.21273/HORTSCI17074-23
The 1-aminocyclopropane-1-carboxylate (ACC) (nmol⋅g−1) (A and B) and malonyl ACC (MACC) (nmol⋅g−1) (C and D) of untreated control and aminoethoxyvinylglycine (AVG)-treated ‘Gala’ apples with absorbance difference index (IAD) values for harvest 1 (H1) of 0.6 to 0.8 (A) and for harvest 2 (H2) of 0.2 to 0.4 (B) kept at 20 °C after harvest or after storage at 0.5 °C for 3 weeks in 2020. The time of fruit harvest or transfer from 0.5 °C to 20 °C is represented as day 0. () Control fruit. () Fruit treated with AVG. () Control fruit (0.5–20 °C). () Fruit treated with AVG (0.5–20 °C). Bars represent ± SE (n = 6), whereas least significant difference values represent P < 0.05.
Citation: HortScience 58, 5; 10.21273/HORTSCI17074-23
High IECs were only found in the control fruit and were enhanced by cold storage (Fig. 4A, B). Cold storage caused an earlier and more rapid increase of IEC in fruit from both IAD categories, but the patterns of change differed. In the 0.6 to 0.8 category, the IEC of cold-stored fruit was higher than that of the treatment without cold storage, except at the final time point (Fig. 4A). In contrast, the IEC of fruit in the 0.2 to 0.4 category increased quickly but was higher than that of the fruit without cold storage until approximately day 10 (Fig. 4B). Cold storage had little effect on the IEC of AVG-treated fruit, with averages of 1.77 μL⋅L−1 and 3.71 μL⋅L−1 in the 0.6 to 0.8 and 0.2 to 0.4 categories, respectively.
Flesh firmness decreased at 20 °C, but the rate of change was affected greatly by AVG and/or cold storage within each IAD category (Fig. 5). Flesh firmness of fruit, either untreated or AVG-treated, at or close to the time of harvest was similar (Fig. 5A, B), but softening of the AVG-treated fruit was slower than that of the control. Cold-stored fruit of both treatments were softer than those without cold storage; control fruit softened faster than AVG-treated fruit, but more consistently so in those of the 0.6 to 0.8 category compared with those of the 0.2 to 0.4 category.
Selected timepoints were used for the ACC and MACC analysis. Cold storage enhanced ACC accumulation in the untreated 0.6 to 0.8 category fruit compared with those without cold storage (Fig. 6A). Even though cold storage enhanced ACC accumulation, patterns of change were different for the 0.2 to 0.4 category, (Fig. 6B). Both ACC and MACC concentrations remained low in the AVG-treated fruits. MACC accumulation in cold-stored 0.6 to 0.8 category fruit showed a trend similar to that found for ACC and similar to the pattern observed in the 0.2 to 0.4 category fruit (Fig. 6C, D).
Discussion
Apple cultivars vary greatly in their responses to cold storage temperatures, with those such as Braeburn, Golden Delicious, Granny Smith, and Lady Williams requiring exposure to chilling temperatures to initiate ethylene production (Jobling and McGlasson 1995; Jobling et al. 1991, 2003; Knee et al. 1983; Tian et al. 2002). These responses are analogous to the cold requirement of winter pears to produce ethylene and ripen properly (Lelievre et al. 1997; Villalobos-Acuna and Mitcham 2008). Previous research of the ‘Royal Gala’ sport found that the fruit did not require exposure to low temperatures to produce ethylene, whereas ethylene production in fruit stored at 1 °C was lower than that at 20 °C; after transfer from 1 °C to 20 °C, ethylene production increased and then decreased before reaching rates similar to that for fruit at only 20 °C (Larrigaudiere et al. 1997). In contrast, during our experiment, although ethylene production occurred without cold exposure of untreated fruit, exposure of fruit to 0.5 °C for 21 d resulted in more rapid and higher ethylene production rates and IEC than for fruit kept at only 20 °C (Figs. 2 and 4). The effect of cold exposure was more like that shown for Granny Smith than for Royal Gala (Larrigaudiere et al. 1997), albeit with a delayed response in those cultivars.
The reason for the differences between the two studies of ‘Gala’ are unclear. The fruit of both IAD categories were preclimacteric at the time of harvest (Figs. 2 and 4); therefore, they were physiologically similar to those observed during the study by Larrigaudiere et al. (1997). Harvest of fruit was described as “optimal” based on firmness and soluble solid concentrations in that study, whereas our harvests spanned commercial harvest dates and, during this experiment, were restricted to IAD values. It seems unlikely that sport type affected the response to cold storage given the similarities in maturation and storage performance (Walsh and Volz 1990). Different harvest times also result in differences in ethylene production, even for the fruit of the same cultivar from a same orchard (Argenta et al. 2021; Bulens et al. 2012). The influence of weather patterns cannot be excluded as a factor, although Gala is an early harvested cultivar and less exposed to temperature variations such as cold nights than later season ones.
The length of time that fruit was stored at cold temperature, 10 d (Larrigaudiere et al. 1997) compared with 21 d in our study, could be a factor. However, longer exposure times were associated with more rapid responses in ‘Braeburn’ and ‘Granny Smith’ apples (Jobling et al. 1991; Tian et al. 2002); therefore, we assume that a greater effect of cold storage would have been detected for 21 d than for shorter times.
IAD values decrease over time as fruit mature and ripen, reflecting loss of chlorophyll (Sadar and Zanella 2019; Toivonen and Hampson 2014; Toivonen and Lannard 2021; Toivonen et al. 2014). The IAD categories of 0.2 to 0.4 and 0.6 to 0.8 in year 1 for the same harvest date and in year 2 for sequential harvests were selected during this study. The assumption was that the more mature fruit (0.2–0.4) would have a shorter delay than the less mature fruit (0.6–0.8) before autocatalytic ethylene production occurred. However, ethylene production occurred earlier in fruit of the 0.6 to 0.8 category (Fig. 2B) than those of the 0.2 to 0.4 category (Fig. 2A). Additionally, the maximum ethylene production rates of fruit in the 0.6 to 0.8 category were higher than those of fruit in the 0.2 to 0.4 category. The patterns of change of IEC (Fig. 4A) were similar to those of ethylene production for the 0.6 to 0.8 category fruit (Fig. 2A). Differences in the IEC between the 20 °C and the cold-stored fruit of the 0.2 to 0.4 category were less clear (Fig. 4B) than those for ethylene production (Fig. 2B). Enhanced IECs because of cold treatments were evident, but they were higher in the control fruit earlier, and IECs in the two treatments overlapped with increasing storage time.
AVG delays ripening of fruit by inhibiting ethylene production, and persistence of this inhibition is dependent on the concentration and timing of application as well as cultivar and season (Arseneault and Cline 2016; Robinson et al. 2009; Schupp and Greene 2004). Treated fruit started to recover from inhibition of ethylene production in year 1 (Fig. 1), but not to any significant extent in year 2 (Figs. 2 and 4). The effect of cold storage on ethylene production of AVG-treated fruit when they recover from inhibition remains unknown because of the persistent fruit response to the plant growth regulator in year 2 compared with year 1.
The respiration rate of a fruit reflects the fruit metabolism as well as its ripening stage; for apples, as a climacteric fruit, it is typically associated with ethylene production. Respiration rates of ‘Gala’ apples were consistently higher in the control fruit at 20 °C after cold storage than in other treatments (Fig. 3). However, respiration rates of cold-treated AVG-treated fruit were often also higher than those of the fruit without cold storage. Those without cold storage were generally low and usually had a greater extent in the AVG-treated fruit than control fruit. The effects of cold treatment on ethylene production and respiration rates were dissociated. The reasons for the differences are interesting and warrant further investigation, but they could represent a “stress” response of fruit to cold storage. No other studies, apart from that by Jobling et al. (1991), have measured respiration (internal CO2 concentrations) by studying cold storage-induced ethylene production; furthermore, they also found that cold storage resulted in differential responses of respiration rates and ethylene production of ‘Granny Smith’ apples.
The effect of short-term cold temperatures on flesh firmness has not been included in studies of cold storage effects on ethylene responses of apple. Flesh firmness decreased during storage of ‘Gala’ fruit in all treatments; although it was lower after cold storage exposure for only 21 d, the rate of softening between the untreated and cold-treated fruit was similar (Fig. 5). Quantifying the effects of cold on apple softening is complicated by the presence of endogenous ethylene because both cold storage and ethylene coordinate the ripening process (Tacken et al. 2010). It is likely that the enhanced softening was directly related to ethylene production (Figs. 2 and 4). However, the AVG-treated fruit, while softening at slower rates than the untreated and cold-exposed fruit, were also less firm after cold storage (Fig. 5). AVG typically slows softening of apple cultivars (Arseneault and Cline 2016), but the reason for the cold storage effects on firmness of both untreated and AVG-treated fruit is unclear. Tacken et al. (2010) used ACO1-suppressed ‘Royal Gala’ apples to investigate the effects of low temperatures and found that cold-treated fruit ripened independently of added ethylene. From a practical point of view, the stimulation of softening in cold-treated fruit without AVG could have significant implications for the maintenance of fruit quality. The effects of short-term temperature exposure and AVG on the triphasic softening patterns of apples (Johnston et al. 2001, 2002) are worthy of further investigation.
Conclusion
Short-term cold storage stimulated a rapid onset of common indicators of ripening. Ethylene production and respiration rates, IEC, fruit softening, and concentrations of ACC and MACC were all enhanced in cold-treated fruit after being transferred to a warm environment. Harvest maturity and preharvest AVG treatment interacted with cold and storage treatment to affect fruit ripening. Fruit in the 0.2 to 0.6 category showed a relatively mild ethylene production, but a more rapid respiration increase compared with fruit in the 0.6 to 0.8 category. Preharvest AVG treatment inhibited ACC accumulation in both cold-treated fruit and untreated control fruit, suppressing cold-induced ethylene production as well as other ripening processes in ‘Gala’ apple. We conclude that advanced fruit maturity and preharvest AVG treatment negatively influence cold-induced ethylene production to different degrees. Other ripening responses including the increasing rates of respiration and fruit softening were enhanced in fruit with advanced fruit maturity, but they were compromised by preharvest AVG treatment.
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