E ﬀ ect of Ethylene-Insensitive Mutation etr2b on Postharvest Chilling Injury in Zucchini Fruit

: Zucchini is a vegetable fruit that is very susceptible to postharvest chilling injury, and fruit ethylene production is correlated with chilling injury sensitivity, such that the more tolerant the cultivar, the lower is its ethylene production. It is expected that zucchini fruit with reduced sensitivity to ethylene would have a higher chilling injury tolerance. In this study, we compared the postharvest fruit quality of wild type and ethylene-insensitive mutant etr2b , in which a mutation was identiﬁed in the coding region of the ethylene receptor gene CpETR2B . Flowers from homozygous WT (wt / wt), mutant plants in homozygous ( etr2b / etr2b ) and heterozygous (wt / etr2b ) were hand-pollinated, and all fruits were harvested with the same length, at about 8 days after pollination. After harvesting, fruit of each genotype was randomly divided in 3 batches of 12 fruits each (four replications with three fruits each), and then stored at 4 ◦ C and 95% RH. At 0, 7, and 14 days after cold storage, each batch was used to assess ethylene production, respiration rate, weight and ﬁrmness loss, chilling injury, and oxidative stress metabolites. The results showed a lower chilling injury associated with lower cold-induced ethylene production in the mutant fruit, in comparison with the WT fruit. These data demonstrated that the ethylene-insensitive etr2b mutant fruit was more tolerant to chilling injury, conﬁrming that basal ethylene in the still undamaged fruit could function as a modulator of post-harvest chilling injury. Moreover, the higher chilling tolerance of the etr2b mutant fruit was not associated with MDA content, but was concomitant with a reduction in the accumulation of hydrogen peroxide in the refrigerated mutant fruit.


Introduction
Cold storage is the most important postharvest technology for fruit and vegetable conservation. However, below the optimal temperature, perishable plant commodities might shorten their shelf life and reduce their postharvest quality, suffering significant chilling injury (CI) symptoms, such as damage on their surface or pitting, and increased weight loss and dehydration.
Zucchini is a vegetable fruit that is very susceptible to CI, and its postharvest storage at temperatures below 8-10 • C rapidly reduces its quality, as a result of considerable loss of water and fruit surface damage. To improve the zucchini's postharvest life and to reduce economic losses, it is essential to and then selfed for their maintenance. All plants were grown in greenhouses under the typical cultural practices for zucchini crops in the experimental field of the University of Almería, Almería, Spain.
Flowers were hand-pollinated, and the fruit harvested with the same length for about 8 days after pollination. After harvesting, fruit of each genotype was randomly divided in four replications of three fruits each, and then stored at 4 • C and 95% RH for 14 days. Four replications with three fruits each were used to assess ethylene production, respiration rate, weight and firmness loss, CI and oxidative stress metabolites in WT, heterozygous and homozygous fruit on days 0, 7, and 14, after cold storage.

Genotyping etr2b Mutation Using TaqMan Probes
Genotyping analysis was performed to identify the homozygous WT (wt/wt), heterozygous (wt/etr2b) and homozygous mutant (etr2b/etr2b) plants from BC2S1 populations. DNA from young leaves of each individual plant was isolated and used for genotyping, by using real-time PCR with TaqMan probes. The multiplex PCRs were done using the SensiFAST™ Probe No-ROX Kit (Meridian Bioscience, Inc. Menphis, TN, USA) a set of forward and reverse primers, amplifying the polymorphic sequence, and two allele-specific probes descriptive of the SNP of interest (C-T). The WT probe was labelled with FAM dye, while the mutant probe was labelled with HEX reporter dye. The BHQ1 quencher molecule was used in both probes. Primers and probes sequences were previously described [15].

Ethylene Production and Respiration Rate
For each replications, 3 fruits were enclosed in 10 L air-tight container for 6 h at 20 • C, then the ethylene content was determined three times, by means a gas chromatograph model Varian 3900GC (Varian analytical Instruments, Walnut Creek, CA, USA), using an FID detector, and the ethylene content was expressed as nL ethylene g −1 FW 6 h −1 .
Respiration rate was assessed by analyzing the CO 2 three time, by means of a Check Mate II Headspace Analysers (Dansensor), and the respiration rate was expressed as ml of CO 2 kg −1 FW 6 h −1 .

Weight Loss, Firmness, and Chilling Injury Index
Twelve individual fruits of each genotype (WT and heterozygous and homozygous) were weighed on days 0, 7, and 14 of cold storage, assessing the percentage of weight loss in each fruit. Fruit firmness was determined by a penetration test in the distal region of the fruits, employing a TA.TX Plus Texture analyzer (Stable Micro-System, Godalming, Surrey, UK). The penetration test was performed by using a 4 mm-diameter probes, with a speed of 2 mm·s −1 and a depth of penetration of 10 mm. Each fruit was punctured three times. The firmness was expressed as maximum force reached (N).

MDA and H 2 O 2 Contents
Malonyldialdheyde (MDA) was determined, following the methodology proposed by Carvajal et al. [1], with some modifications [4]. Exocarp was homogenized in 1:10 (w/v) trichloroacetic acid (TCA) and 0.25% (w/v) of a solution of 2-thiobarbituric acid (TBA) 1:10 (w/v), and then heated at 95 • C in a water bath for 30 min, followed by immediate cooling in ice and centrifugation at 4000× g, for 20 min at 4 • C. The absorbance of the supernatant was measured at 532 and 600 nm. MDA content was expressed as nmol MDA g −1 FW. H 2 O 2 content was determined by using the procedure in Alexieva et al. [17], with minor modifications. The exocarp was homogenized in TCA and cold centrifugated, then 0.5 mL supernatant was added with a potassium buffer (pH 7) and potassium iodide. The mixture was kept in the dark for 60 min, after which, the absorbance at 390 nm was measured. H 2 O 2 content was expressed as µmol H 2 O 2 g −1 FW. All reagents and chemicals were obtained from Sigma-Aldrich, Madrid, Spain.

Statistical Analysis
For statistical analysis, STATGRAPHICS ® Centurion XVI (Statpoint Technologies, Inc., Warrenton, VA, USA) was used. The data were treated by ANOVA, followed by a least significant difference test with a significance level of 95%. A Kolmogorov-Smirnov test was used to check the normality of data distribution. When normality failed, the variables were transformed and a non-parametric Kruskal-Wallis test at <0.05 significance level was used. Figure 1 shows the effect of the ethylene-insensitive mutation etr2b on fruit phenotype, at harvest and after 14 days of cold storage. The mutation enables the flower to retain its green color and to be attached to the fruit at harvest, indicating that the reduced ethylene sensitivity of the etr2/etr2 mutant delays floral organ abscission. After cold storage, the etr2/etr2b flowers gradually dries up and senesces, but the fruits remain fresher and show much less CI than the WT fruit.

Response of Zucchini Ethylene-Insensitive Mutant Fruit to Cold Storage
Agriculture 2020, 10, x FOR PEER REVIEW 4 of 14 H2O2 content was determined by using the procedure in Alexieva et al. [17], with minor modifications. The exocarp was homogenized in TCA and cold centrifugated, then 0.5 mL supernatant was added with a potassium buffer (pH7) and potassium iodide. The mixture was kept in the dark for 60 min, after which, the absorbance at 390 nm was measured. H2O2 content was expressed as μmol H2O2 g −1 FW. All reagents and chemicals were obtained from Sigma-Aldrich, Madrid, Spain.

Statistical Analysis
For statistical analysis, STATGRAPHICS ® Centurion XVI (Statpoint Technologies, Inc., Warrenton, VA, USA) was used. The data were treated by ANOVA, followed by a least significant difference test with a significance level of 95%. A Kolmogorov-Smirnov test was used to check the normality of data distribution. When normality failed, the variables were transformed and a nonparametric Kruskal-Wallis test at <0.05 significance level was used. Figure 1 shows the effect of the ethylene-insensitive mutation etr2b on fruit phenotype, at harvest and after 14 days of cold storage. The mutation enables the flower to retain its green color and to be attached to the fruit at harvest, indicating that the reduced ethylene sensitivity of the etr2/etr2 mutant delays floral organ abscission. After cold storage, the etr2/etr2b flowers gradually dries up and senesces, but the fruits remain fresher and show much less CI than the WT fruit. The effects of the ethylene-insensitive mutation on different postharvest fruit-quality parameters are shown in Figure 2. Cold storage at 4 • C induced pitting on the surface of zucchini fruits, with a noticeable increase in the CI index, after 14 days of storage ( Figure 1). However, cold storage affected the WT and mutant fruits differentially. On the 7th day of storage, the WT fruit was more sensitive to cold than the heterozygous and homozygous etr2b fruits. On day 14 of storage, however, only the homozygous mutant fruits etr2b/etr2b reduced the CI index, compared to the WT and heterozygous fruits ( Figure 1). This alleviation of CI was not accompanied by a reduced weight loss or firmness, showing that the WT and etr2b fruits had statistically similar values for these two parameters (Figure 1). At the time of harvest, the heterozygous fruits presented a slightly lower value of firmness, but as the storage time went by, the differences in firmness between the fruits of the different genotypes were reduced, until no differences were found after 14 days of cold storage ( Figure 1).

Response of Zucchini Ethylene-Insensitive Mutant Fruit to Cold Storage
Agriculture 2020, 10, x FOR PEER REVIEW 5 of 14 Figure 1. General appearance of WT and the etr2b/etr2b fruit at harvest and after 14 days of storage, at 4 °C. Note that the mutant fruit maintain the flower attached at harvest. After cold storage, the flower is senescent but the fruit have a reduced percentage CI.
The effects of the ethylene-insensitive mutation on different postharvest fruit-quality parameters are shown in Figure 2. Cold storage at 4 °C induced pitting on the surface of zucchini fruits, with a noticeable increase in the CI index, after 14 days of storage ( Figure 1). However, cold storage affected the WT and mutant fruits differentially. On the 7th day of storage, the WT fruit was more sensitive to cold than the heterozygous and homozygous etr2b fruits. On day 14 of storage, however, only the homozygous mutant fruits etr2b/etr2b reduced the CI index, compared to the WT and heterozygous fruits ( Figure 1). This alleviation of CI was not accompanied by a reduced weight loss or firmness, showing that the WT and etr2b fruits had statistically similar values for these two parameters ( Figure  1). At the time of harvest, the heterozygous fruits presented a slightly lower value of firmness, but as the storage time went by, the differences in firmness between the fruits of the different genotypes were reduced, until no differences were found after 14 days of cold storage (Figure 1 Figure 3 compares the pattern of ethylene production and respiration rate of the WT and etr2b fruits, throughout cold storage. The ethylene production profile was in agreement with what was previously observed in the zucchini fruit. At harvest time, ethylene production was very low and did not show statistical differences between WT and the heterozygous and homozygous etr2b plants. After 7 days of cold storage and a rewarming period of 6 h, a burst of ethylene was found to be more significant in the WT than in the mutant fruit. After 2 weeks of cold storage, ethylene production dropped sharply to levels almost as low as those achieved at harvest time ( Figure 3). The ethylene-insensitive mutation significantly reduced cold-induced ethylene on day 7 of storage, both in the heterozygous and homozygous conditions ( Figure 3).

Effect of Ethylene-Insensitive Mutations on Ethylene and CO 2 Production in Cold-Stored Zucchini Fruit
No statistical differences were found between the respiration rate of WT and etr mutant fruit on day 0, 7, and 14 of cold storage, indicating that a reduction in ethylene production and sensitivity under cold storage was not able to alter the fruit's respiration pattern in terms of the respiration rate.
Agriculture 2020, 10, x FOR PEER REVIEW 7 of 14 Figure 3 compares the pattern of ethylene production and respiration rate of the WT and etr2b fruits, throughout cold storage. The ethylene production profile was in agreement with what was previously observed in the zucchini fruit. At harvest time, ethylene production was very low and did not show statistical differences between WT and the heterozygous and homozygous etr2b plants. After 7 days of cold storage and a rewarming period of 6 h, a burst of ethylene was found to be more significant in the WT than in the mutant fruit. After 2 weeks of cold storage, ethylene production dropped sharply to levels almost as low as those achieved at harvest time ( Figure 3). The ethyleneinsensitive mutation significantly reduced cold-induced ethylene on day 7 of storage, both in the heterozygous and homozygous conditions ( Figure 3).

Effect of Ethylene-Insensitive Mutations on Ethylene and CO2 Production in Cold-Stored Zucchini Fruit
No statistical differences were found between the respiration rate of WT and etr mutant fruit on day 0, 7, and 14 of cold storage, indicating that a reduction in ethylene production and sensitivity under cold storage was not able to alter the fruit's respiration pattern in terms of the respiration rate.

Effect of Ethylene-Insensitive Mutations on Oxidative Stress
We previously observed that CI tolerance in Zucchini fruit was associated with a reduction in oxidative stress. Given that MDA is caused by lipid peroxidation, and is therefore an indicator of cell membrane integrity, we compared the MDA content in the exocarp of WT and etr2b fruits (Figure 4). During cold storage, MDA increased in the WT and heterozygous and homozygous mutant fruits, but the content of MDA in the mutant and WT fruits was similar.
The content of H 2 O 2 in the fruit exocarp of the different genotypes was also very similar throughout the storage period, but after 7 days of cold storage, statistical differences were found between the H 2 O 2 content of the WT and etr2b mutant fruit. The reduction in ethylene production after 7 days of cold storage in the homozygous etr2b/etr2b fruit, was therefore associated with a diminution of the of H 2 O 2 content ( Figure 4). As expected, the heterozygous wt/etr2b fruit, whose plants have an intermediate sensitivity to ethylene [14,15], showed an intermediate content of H 2 O 2 at 7 day of cold storage, with respect to the WT and the homozygous mutant fruits (Figure 4).

Effect of Ethylene-Insensitive Mutations on Oxidative Stress
We previously observed that CI tolerance in Zucchini fruit was associated with a reduction in oxidative stress. Given that MDA is caused by lipid peroxidation, and is therefore an indicator of cell membrane integrity, we compared the MDA content in the exocarp of WT and etr2b fruits (Figure 4). During cold storage, MDA increased in the WT and heterozygous and homozygous mutant fruits, but the content of MDA in the mutant and WT fruits was similar.
The content of H2O2 in the fruit exocarp of the different genotypes was also very similar throughout the storage period, but after 7 days of cold storage, statistical differences were found between the H2O2 content of the WT and etr2b mutant fruit. The reduction in ethylene production after 7 days of cold storage in the homozygous etr2b/etr2b fruit, was therefore associated with a diminution of the of H2O2 content ( Figure 4). As expected, the heterozygous wt/etr2b fruit, whose plants have an intermediate sensitivity to ethylene [14,15], showed an intermediate content of H2O2 at 7 day of cold storage, with respect to the WT and the homozygous mutant fruits (Figure 4).

Discussion
During cold storage, zucchini showed significant CI symptoms, the most important of which are pitting and weight loss [4,16]. The relationship between the incidence of pitting and increased weight loss found in this and other works might indicate that surface damage and evapotranspiration are both dependent on cold storage time [18]. Moreover, pitting damage caused by cold in zucchini surface can affect membrane integrity, favoring greater weight loss [8,19]. Similar water loss in WT and ethylene-insensitive etr2b fruits could be due to the presence of floral organs in the etr2b mutant fruit (Figure 1). The loss of water associated with the senescence of the petals in the mutant fruit with the attached flower, could cause this to lose the same water as the WT fruit, despite the former being more cold-tolerant. On the other hand, zucchini is very sensitive to cold storage, but fruit postharvest firmness was hardly affected in the WT and the etr2b fruits. It was reported that zucchini firmness was more dependent on the cultivar than on other factors, such as cold damage or weight loss [1,2]. The reduced metabolism and the lower activity of cell wall degrading enzymes, such as polygalacturonase (PG) and pectin methylesterase (PME) under cold storage [20][21][22], might account for the maintenance of zucchini fruit firmness in our experiments.
Zucchini is a non-climacteric fruit with a very low ethylene production at harvest and during its storage period at 20 °C [16]. When the fruit is stored cold for 7 days, and then transferred to room temperature for a few hours, it produces a burst of ethylene. This cold-induced ethylene disappears when cold storage continues up to 14 days, before rewarming [2,16]. A clear association was found between this cold-induced ethylene and CI susceptibility in zucchini fruit, since the fruits of cultivars and plants from segregating populations that are CI-tolerant, always produce less ethylene than those that are more susceptible to CI [5] and the treatments that lessen CI in zucchini, including individual shrink wrapping and 1-MCP, concomitantly reducing the production of cold-induced ethylene on day 7 of cold storage [2,4]. In this study, we also found that reduced CI in the ethylene-insensitive etr2b fruit compared to the WT fruit on days 7 and 14 of cold storage, was also correlated with a reduced production of cold-induced ethylene. The production of ethylene is also induced after chilling storage and rewarming in a number of non-climacteric fruits, such as cucumber, citrus, pear, and kiwi [23][24][25][26]. However, this cold-induced ethylene is not required in zucchini for the onset of CI in zucchini squash, since it was demonstrated that CI appears in the refrigerated chamber before rewarming of the fruit at room temperature, and therefore, before the burst of ethylene production [2,16]. Accordingly, the cold-induced ethylene is actually the fruit's response to cold damage, and the lower production of this cold-induced ethylene in etr2b mutants might indicate that their fruit suffers less cold damage than that of WT, and is therefore, more tolerant to CI.

Discussion
During cold storage, zucchini showed significant CI symptoms, the most important of which are pitting and weight loss [4,16]. The relationship between the incidence of pitting and increased weight loss found in this and other works might indicate that surface damage and evapotranspiration are both dependent on cold storage time [18]. Moreover, pitting damage caused by cold in zucchini surface can affect membrane integrity, favoring greater weight loss [8,19]. Similar water loss in WT and ethylene-insensitive etr2b fruits could be due to the presence of floral organs in the etr2b mutant fruit (Figure 1). The loss of water associated with the senescence of the petals in the mutant fruit with the attached flower, could cause this to lose the same water as the WT fruit, despite the former being more cold-tolerant. On the other hand, zucchini is very sensitive to cold storage, but fruit postharvest firmness was hardly affected in the WT and the etr2b fruits. It was reported that zucchini firmness was more dependent on the cultivar than on other factors, such as cold damage or weight loss [1,2]. The reduced metabolism and the lower activity of cell wall degrading enzymes, such as polygalacturonase (PG) and pectin methylesterase (PME) under cold storage [20][21][22], might account for the maintenance of zucchini fruit firmness in our experiments.
Zucchini is a non-climacteric fruit with a very low ethylene production at harvest and during its storage period at 20 • C [16]. When the fruit is stored cold for 7 days, and then transferred to room temperature for a few hours, it produces a burst of ethylene. This cold-induced ethylene disappears when cold storage continues up to 14 days, before rewarming [2,16]. A clear association was found between this cold-induced ethylene and CI susceptibility in zucchini fruit, since the fruits of cultivars and plants from segregating populations that are CI-tolerant, always produce less ethylene than those that are more susceptible to CI [5] and the treatments that lessen CI in zucchini, including individual shrink wrapping and 1-MCP, concomitantly reducing the production of cold-induced ethylene on day 7 of cold storage [2,4]. In this study, we also found that reduced CI in the ethylene-insensitive etr2b fruit compared to the WT fruit on days 7 and 14 of cold storage, was also correlated with a reduced production of cold-induced ethylene. The production of ethylene is also induced after chilling storage and rewarming in a number of non-climacteric fruits, such as cucumber, citrus, pear, and kiwi [23][24][25][26]. However, this cold-induced ethylene is not required in zucchini for the onset of CI in zucchini squash, since it was demonstrated that CI appears in the refrigerated chamber before rewarming of the fruit at room temperature, and therefore, before the burst of ethylene production [2,16]. Accordingly, the cold-induced ethylene is actually the fruit's response to cold damage, and the lower production of Agriculture 2020, 10, 532 9 of 12 this cold-induced ethylene in etr2b mutants might indicate that their fruit suffers less cold damage than that of WT, and is therefore, more tolerant to CI.
It was suggested that ethylene plays an essential role in CI development [27,28]. In fact, it is long known that methods that induce a reduction in ethylene synthesis leads to an improvement in cold damage. Thus, ACO-anti-sense melons showed a higher tolerance to cold damage than WT, when kept at 2 • C [29]. In the opposite direction and even more interestingly, Obando et al. [30] observed that crossing two non-climacteric lines of melons obtained a line of climacteric melons, and that this character was associated with a higher sensitivity to chilling damage. In zucchini, the burst of cold-induced ethylene is not required for the onset of CI, as mentioned above [2,5,16], but ethylene must be involved in a positive regulation of CI. Thus, we previously found that ethylene is slightly induced in refrigerated fruit before rewarming [2]. Moreover, external treatments with the ethylene inhibitor 1-MCP reduced CI in the fruit of cold-susceptible zucchini cultivars [11]. Here, we demonstrated that the mutation of ethylene receptor CpETR2B, leading to ethylene insensitivity [14,15], are also able to reduce postharvest CI, concomitantly with a reduction of ethylene ( Figure 3). In cold-sensitive grapefruit, an induction of certain ethylene receptor isoforms (ETRs) was observed, as well as ethylene response factors (ERFs), suggesting a close relationship between ethylene perception and signaling with cold damage [28,31]. Huang et al. [32] found that the levels of expression of ethylene signal transduction genes PgERF1 and PgERF2 in pomegranate, correlated with increased cold damage. However, no clear linear relationship was found between PgETR expression and chilling injury. The fact that the inhibition of ethylene response in the etr2b mutants was able to reduce CI in zucchini also indicates an active role of ethylene in the control of cold damage. Given that 1-MCP only alleviate CI in the most cold-susceptible cultivars, and that the reduction of CI in the etr2b mutant is not very pronounced, it is likely that other cooperating factors participate together with ethylene, in the regulation of fruit chilling response in zucchini squash. The effect of the individual mutation etr2b on CI is even smaller than that obtained by 1-MCP treatments [2], because 1-MCP would surely affect all ethylene receptors in the fruit, while the individual ethylene insensitivity etr2b mutation would only affect just one specific ethylene receptor, resulting in 43.5% and 63.7% of ethylene insensitivity in wt/etr2b and etr2b/etr2b, respectively [15].
CI alleviation in the etr2b mutant fruit was not associated with a reduction in the respiration rate, but significantly reduced the accumulation of oxidative stress metabolites such as hydrogen peroxide. Several postharvest treatments that alleviate CI symptoms in zucchini reduce the fruit respiration rate and oxidative stress [33,34]. The reported effect of 1-MCP on the respiration rate of refrigerated fruit was variable, and only the fruit of more susceptible zucchini cultivars was able to reduce its respiration [2]. Moreover, the onset of CI in refrigerated non-climacteric fruit is concomitant with an increase in the production of Reactive Oxygen Species (ROS) and oxidative damage of cell membranes and MDA accumulation [4]. It was demonstrated that cold tolerance of the zucchini fruit is associated with a reduction in MDA content [4,8]. The fact that the etr2b mutant fruit showed reduced CI symptoms and H 2 O 2 content, but accumulated a similar level of MDA than WT fruit (Figure 4) could indicate that the role of ethylene signaling in lipid peroxidation is limited. The role of ethylene receptors and signaling components in oxidative stress is not clear. In Arabidopsis, it was demonstrated that the ethylene-insensitive ein2-1 mutation was able to alleviate plant oxidative stress [35]. In four ethylene-insensitive mutants of tobacco plants, however, an increased MDA content was found when the plants were undergoing oxidative stress, in response to drought [36], and in the ethylene-insensitive mutant Never ripe of tomato, plants showed an enhanced MDA content in roots and leaves, in response to NaCl and CdCl 2 stress [37].

Conclusions
Comparison of postharvest quality of WT and etr2b mutant fruit during cold storage showed that ethylene could modulate cold damage in zucchini vegetable fruit. The mutation was able to reduce plant ethylene sensitivity, concomitantly, with a reduced production of cold-induced ethylene, and an enhanced chilling tolerance of refrigerated fruit. The ethylene-insensitive mutation also reduced the accumulation of hydrogen peroxide, but had no effect on the MDA content.

Funding:
This research was funded by grants AGL2014-54598-C2-1-R, AGL2017-82885-C2-1-R, and UAL18-BIO-B017B, which were partly funded by the European Regional Development Fund and partly by the Spanish Ministry of Science and the University of Almería. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.