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Smart Glass Film Reduced Ascorbic Acid in Red and Orange Capsicum Fruit Cultivars without Impacting Shelf Life

Smart Glass Film (SGF) is a glasshouse covering material designed to permit 80% transmission of photosynthetically active light and block heat-generating solar energy. SGF can reduce crop water and nutrient consumption and improve glasshouse energy use efficiency yet can reduce crop yield. The effect of SGF on the postharvest shelf life of fruits remains unknown. Two capsicum varieties, Red (Gina) and Orange (O06614), were cultivated within a glasshouse covered in SGF to assess fruit quality and shelf life during the winter season. SGF reduced cuticle thickness in the Red cultivar (5%) and decreased ascorbic acid in both cultivars (9–14%) without altering the overall morphology of the mature fruits. The ratio of total soluble solids (TSSs) to titratable acidity (TA) was significantly higher in Red (29%) and Orange (89%) cultivars grown under SGF. The Red fruits had a thicker cuticle that reduced water loss and extended shelf life when compared to the Orange fruits, yet neither water loss nor firmness were impacted by SGF. Reducing the storage temperature to 2 °C and increasing relative humidity to 90% extended the shelf life in both cultivars without evidence of chilling injury. In summary, SGF had minimal impact on fruit development and postharvest traits and did not compromise the shelf life of mature fruits. SGF provides a promising technology to block heat-generating solar radiation energy without affecting fruit ripening and marketable quality of capsicum fruits grown during the winter season.


Introduction
Rising populations and crop nutrient deficiency are substantial worldwide issues that require sustainable solutions [1][2][3]. Protected cropping facilities provide precise environmental control to promote year-round high yielding crop production [4], yet energy consumption can be a limiting factor [5]. Recent advances in innovative covering material technologies can reduce glasshouse energy consumption while transmitting sufficient light to maintain crop production [6,7]. While most studies evaluating covering materials for greenhouse crop production have focused on reporting crop growth and yield, few studies have investigated the impact of cover materials on quality. For example, covering materials tested on greenhouse production of tomato [8], cucumber [9] and lettuce [10] displayed variable effects on fruit development and quality formation among cultivars depending   (15 to 65 DAP). Line graphs display fruit weight (c-e), length to diameter ratio (L/D; f-g), length (h), and pericarp thickness (i-k). Mean values and standard error bars (n = 12 fruits from two experimental bays) are displayed. Bar plots depict the average of fruit weight (e), length (h), and thickness of pericarp (k) of Red and Orange fruits at the mature stage of development (65 DAP). Horizontal lines and stars denote statistical significance analyzed by one-way ANOVA (*: p ≤ 0.05, **: p ≤ 0.01, and ***: p ≤ 0.001). Abbreviations: Smart Glass Film (SGF); days after pollination (DAP). Table 1. Effects of SGF and cultivar interactions on morphology and postharvest quality traits from Red and Orange mature ripe fruits (65 DAP). Data represents the average from multiple fruits (n = 12) and the standard error of mean is displayed in brackets. Statistical analyses were performed using a one-way ANOVA (*: p ≤ 0.05, **: p ≤ 0.01, and ***: p ≤ 0.001). Abbreviations: Not significant (NS), smart glass film (SGF), percentage difference (%), cultivar (CV), days after pollination (DAP), total soluble solids (TSS), titratable acids (TA), chroma (C*), hue angle (h), lightness (L*), red/green value (a*), yellow/blue value (b*), fresh weight (FW). During early fruit development (seven DAP), a thin cuticle was formed in the fruit exocarp layer that extended to the anticlinal peg (AP) at 15 DAP (Figure 2a-b). Thereafter, cuticle deposition occurred around epidermal cells (25 DAP), leading to a rapid increase in thickness, while sub-epidermal deposition (SD) continued in the anticlinal plane from 45-65 DAP (Figure 2c-g). Cuticular deposition increased with fruit development, reaching a maximum thickness by 35-45 DAP (Figure 2h-i). Red fruits showed a 17.3% thicker cuticle compared to Orange fruits ( Figure 2j; Table 1). The SGF significantly reduced cuticular thickness in Red fruits from 35 to 65 DAP (>5.6%), while in Orange fruits a 13.4% reduction was only observed at 55 DAP (Figure 2h-j). Therefore, the SGF caused a modest reduction in cuticle thickness of mature harvestable Red fruits (65 DAP), which had a much thicker cuticle compared to Orange fruits. (Figure 2c-g). Cuticular deposition increased with fruit development, reaching a maximum thickness by 35-45 DAP (Figure 2h-i). Red fruits showed a 17.3% thicker cuticle compared to Orange fruits ( Figure 2j; Table 1). The SGF significantly reduced cuticular thickness in Red fruits from 35 to 65 DAP (>5.6%), while in Orange fruits a 13.4% reduction was only observed at 55 DAP (Figure 2h-j). Therefore, the SGF caused a modest reduction in cuticle thickness of mature harvestable Red fruits (65 DAP), which had a much thicker cuticle compared to Orange fruits.

SGF Significatly Reduced Ascorbic Acid Content and Caused Subtle Effects on Fruit Quality Indexes
SGF decreased ascorbic acid levels by 8.7% and 14% in both Red and Orange mature fruits (65 DAP), respectively ( Figure 3b). SGF increased TSS in orange fruits by 4.4% and yet decreased titratable acidity by 21% in Red fruits (Table 1). Consequently, SGF significantly increased the TSS/TA ratio in Orange (+89%) more than Red (+29%) fruits ( Figure 3a; Table 1). There was a significant decrease in moisture content (0.3%), increase in ash (6.5%), and reduction in colour parameters (a* 12%, b* 13%, and C* 12.5%) specific to Orange fruits (Table 1). SGF marginally increased pH in Red fruits. The SGF treatment did not affect total carotenoid content and fruit firmness in either cultivar. Therefore, SGF significantly reduced ascorbic acid levels in both varieties and had subtle effects on other fruit quality parameters in a cultivar-dependent manner.
x FOR PEER REVIEW 6 of 15 Principal component analysis (PCA) of quality indexes revealed cultivar-specific differences in moisture, pH, ash, TA, ascorbic acid, and firmness ( Figure 3c). Two primary axes of principal component (PC) variation cumulatively explained 45.2% of the variation. PC1 explained 24.7% of total variation that was positively associated with traits related to moisture, firmness, and fruit weight, and yet negatively associated with ascorbic acid. PC2 explained 20.5% of total variation associating with TSS, ash, pH, and titratable acidity. The Red cultivar was positively related to moisture, weight, and firmness, while the Orange cultivar was correlated with ascorbic acid. Fruit moisture significantly correlated with firmness and fruit weight. Overall, there is a greater separation of quality index traits by cultivar rather than SGF.
The relationship between altered photosynthetically active radiation (PAR; from 45 DAP to 65 DAP) and fruit quality traits were assessed using the Pearson's correlation coefficient (Table 2). Red fruits showed a significant negative linear correlation relationship between PAR and colour parameters that was not observed for Orange fruits ( Table 2). The SGF-related decrease in ascorbic acid in Orange fruits was also significantly correlated to PAR. Overall, the altered light transmittance by SGF led to significant correlation relationships between PAR and fruit colour (Red) or between PAR and ascorbic acid (Orange) in a cultivar-dependent manner. Principal component analysis (PCA) of quality indexes revealed cultivar-specific differences in moisture, pH, ash, TA, ascorbic acid, and firmness ( Figure 3c). Two primary axes of principal component (PC) variation cumulatively explained 45.2% of the variation. PC1 explained 24.7% of total variation that was positively associated with traits related to moisture, firmness, and fruit weight, and yet negatively associated with ascorbic acid. PC2 explained 20.5% of total variation associating with TSS, ash, pH, and titratable acidity. The Red cultivar was positively related to moisture, weight, and firmness, while the Orange cultivar was correlated with ascorbic acid. Fruit moisture significantly correlated with firmness and fruit weight. Overall, there is a greater separation of quality index traits by cultivar rather than SGF.
The relationship between altered photosynthetically active radiation (PAR; from 45 DAP to 65 DAP) and fruit quality traits were assessed using the Pearson's correlation coefficient (Table 2). Red fruits showed a significant negative linear correlation relationship between PAR and colour parameters that was not observed for Orange fruits ( Table 2). The SGF-related decrease in ascorbic acid in Orange fruits was also significantly correlated to PAR. Overall, the altered light transmittance by SGF led to significant correlation relationships between PAR and fruit colour (Red) or between PAR and ascorbic acid (Orange) in a cultivar-dependent manner. Table 2. Pearson's correlation coefficient analysis of photosynthetically active radiation (45 to 65 DAP) with fruit quality indexes in mature ripe fruit (65 DAP). The correlation coefficient and probability values were analyzed by R using "Hmisc" package. Data was analysed from multiple fruits (n = 12). Statistical analysis was performed using a one-way ANOVA (*: p ≤ 0.05 and **: p ≤ 0.01). Abbreviations: Smart glass film (SGF), days after pollination (DAP), total soluble solids (TSS), titratable acids (TA), photosynthetically active radiation (PAR), chroma (C*), hue angle (h), lightness (L*), red/green value (a*), and yellow/blue value (b*).

SGF Did Not Impact the Shelf Life or Fruit Quality
SGF did not significantly impact the fruit water loss in either cultivar when fruits were stored at 20 • C and 30% relative humidity (RH, Figure 4a). Within the first three days of storage, the relative water loss from Red fruits (1.1%) was lower compared to Orange fruits (4.8%), which began to display signs of shriveling. Over 30 days, the total water loss was greater in Orange (−40%) compared to Red (−34%) fruits. There were differences in firmness between the two cultivars that were unaffected by SGF (Figure 4c-e). During 15 days of storage at 20 • C (30% RH), the firmness of Red and Orange fruits decreased by 81% (from 10.4 N to 2.0 N) and 82% (from 6.6 N to 1.2 N), respectively (Figure 4e). The industrial postharvest management practice to store capsicum fruits at 7 • C (90% RH) enhanced firmness of Red fruits, while decreasing temperature to 2 • C (90% RH) enhanced firmness of both fruit cultivars without signs of chilling injury (Figure 4b). Hence, an extended postharvest cooler temperature combined with higher relative humidity can enhance the firmness of Red and Orange fruits. Overall, the differences in shelf-life traits defined by fruit water loss and firmness were cultivar-dependent and not altered by SGF.
The principal component analysis of shelf-life parameters revealed links between the cultivar and impact of SGF. PC1 explained 37.2% of total variation (Figure 4f), which was positively associated with traits related to initial water loss in the first three days of postharvest storage. PC2 explained 18.2% of total variation and was positively associated with the initial three days of loss in firmness, but was negatively related to fruit weight. Red fruits trended towards greater firmness, higher moisture content, and a thicker cuticle deposition. Orange fruits trended towards faster water loss and reduced firmness quicker with a thinner cuticle. Therefore, the shelf life of fruits was largly unaffected by SGF and was cultivar dependent. 81% (from 10.4 N to 2.0 N) and 82% (from 6.6 N to 1.2 N), respectively (Figure 4e). The industrial postharvest management practice to store capsicum fruits at 7 °C (90% RH) enhanced firmness of Red fruits, while decreasing temperature to 2 °C (90% RH) enhanced firmness of both fruit cultivars without signs of chilling injury (Figure 4b). Hence, an extended postharvest cooler temperature combined with higher relative humidity can enhance the firmness of Red and Orange fruits. Overall, the differences in shelf-life traits defined by fruit water loss and firmness were cultivar-dependent and not altered by SGF.

Discussion
New glasshouse covering materials such as SGF alter the light quantity and quality within the growth environment, thereby potentially affecting crop physiology, growth, and yield [7,13,38]. However, it was unknown whether SGF could also affect the development, ripening, and postharvest quality traits of mature fruit from different pigmented cultivars. The current study demonstrates that the altered light in SGF did not impact the development and ripening of Red and Orange fruits cultivated during the winter season. The effects of SGF on fruit quality parameters (e.g., colour, cuticle thickness, moisture content, ash, TSS, TA, and pH) were largely cultivar specific, and subtle changes in colour, TSS/TA, and ascorbic acid were within the range of marketable acceptance. We showed that SGF does not affect fruit water loss, firmness, or postharvest shelf life, revealing that the economic value of the fresh capsicum produce is maintained during storage.

SGF Does Not Affect Fruit Development, Ripening, or Appearance
Fruit development and ripening are important for early yield, and these marketable features are usually genetically and environmentally controlled [20,38]. During early stages of fruit development, additional red and far-red light promotes tomato fruit growth [20,39], and a reduction of PAR by covering materials can negatively affect the commercial fruit size of tomato fruits [21,22]. However, the decrease in PAR and previously reported decrease in red and far-red light caused by SGF [13], did not affect Red or Orange fruit morphology or ripening in this study. Rather, we found differences in fruit size, weight, and pericarp thickness during fruit maturation that were cultivar dependent. The thicker pericarp of Red fruits was associated with increased weight, which was consistent with a previous study [40]. There were subtle fluctuations in developmental parameters (e.g., weight, L/D) of fruits grown under SGF, which were not consistently observed during the 65 days of fruit development. Small differences in marketable fruit size may appear across a season [41], Carotenoid pigments provide Red and Orange capsicum fruits with their visual appeal and reflect the maturity of ripened fruits. Since capsicum fruit are non-climacteric and picked ripe, fruit colour does not usually change after harvest [42]. Carotenoid biosynthesis can be regulated by the light environment leading to changes in fruit colour [28]. It has been shown that carotenoid metabolism in orange and red varieties was responsive to changes in red, blue, and UV light spectrums [29,43,44]. While SGF did not affect the total carotenoid content in either variety, some colour parameters (e.g., L*, a*, b*, C* and h) were slightly lower in Orange fruits. This could be due to changes in individual carotenoid pigments that contribute to the colour variations in capsicum fruit cultivars [29,44]. Therefore, SGF does not appear to affect the marketable appearance of fruits from Red and Orange cultivars.

SGF Reduced Cuticular Deposition in the Red Cultivar
Cuticle deposition of the tomato fruit epidermis helps to protect inner mesocarp tissues from water loss [45,46]. Exposure of tomato fruits to different light conditions can also lead to variations in cuticle thickness [45,46]. We found that the process of cuticle development in capsicum was similar to tomato [46], in that during the early stage of capsicum development the cuticle is thinner and becomes thicker during ripening with an increase in sub-epidermal deposition. Cuticle thickness increased in Red and Orange capsicum fruits in a similar manner throughout development, yet SGF reduced cuticle deposition in Red fruits, indicating that light affects cuticle development in a cultivarspecific manner. UV radiation was positively correlated with cuticle deposition in tomato and sweet cherry fruits [46,47]. SGF was previously shown to reduce UV light transmission to an eggplant crop grown over both summer and winter seasons [13], and this could be the cause of the pronounced decrease in cuticular thickness 35 DAP in Red fruits. The decreased cuticle thickness in Red fruits grown under SGF did not impact water loss and hence postharvest self-life quality. Cuticle thickness is not a conventionally detectable parameter defining the marketability of fresh industrial capsicum produce [48], but may provide insight into shelf life.

SGF Reduced Ascorbic Acid Content
Lower ascorbic acid levels have been reported in tomato fruits and other fresh produce in growth environments with low UV light or that are shaded [49][50][51]. The blockage of 85% UV-B and/or 19% PAR by SGF [13] caused a notable decrease in ascorbic acid content in both Red and Orange capsicum fruits. There was a correlation between PAR and ascorbic acid content in Orange capsicum fruits, and similar cultivar specific effects have been reported for other fruit types [50,52,53]. Indeed, we found that fruit quality index traits were dependent on cultivar, rather than SGF. The accumulation of ascorbic acid provides a measure of the state of ripening as it can be correlated with the accumulation of cell wall polysaccharides and softening of the fruit pericarp [35,54]. However, SGF did not alter capsicum fruit ripening, despite the subtle reduction in ascorbic acid. The Red and Orange capsicum fruit cultivars grown under SGF provide an acceptable level of ascorbic acid (40~45 mg) that can be consumed as a recommended dietary allowance [55].
TSS can vary in fruits grown under different covering materials [7]. Altered far-red and infrared light transmission can affect sugar content differently depending upon the fruit crop. For example, a higher sugar content was observed in melon fruits grown under near infrared ray reducing nets [56], while additional far-red light improved sugar content in tomato fruits [39]. Reductions in PAR caused by flexible photovoltaic rooftop panels in tomato [21] and by SGF in eggplant [13] reduced TSS. We detected subtle changes in TSS and TA that were cultivar dependent. Overall, it was not surprising that the TSS/TA ratio was slightly higher in both Red and Orange capsicum fruit varieties given that SGF lowered ascorbic acid levels. The TSS/TA ratio reflects a taste sensory parameter used to determine fresh produce quality, but the slightly higher TS/TA ratio of capsicum fruits grown under SGF may not be sufficient to affect taste sensory preferences.

Fruit Firmness, Water Loss, and Shelf Life Storage Were Not Affected by SGF
The cuticle limits water loss through the fruit pericarp and capsicum fruits do not contain stomata on the epidermal surface [40,46]. We demonstrated that Red capsicum fruits have a thicker cuticle compared to the Orange variety, as previously reported [30,57], and further showed that this helps to retain water and extend fruit shelf life. While SGF reduced the cuticular thickness in Red fruits, the water loss during storage declined in a manner similar to fruits produced under the control glass. Capsicum fruits have a waxy surface that helps to prevent excess water loss [31,57]. There was a good correlation between fruit firmness, cuticle thickness, and higher moisture content in both fruit types. These traits determine how growers and distributors manage the postharvest storage environment to ensure high fresh produce quality for retail [30]. A higher relative humidity and lower temperature is commonly used to enhance the storage of green bell pepper fruits [58]. SGF did not impact fruit shelf life, nor the firmness and quality of Red and Orange fruits during long-term storage. We conclude that SGF does not have any major effects on the postharvest quality of fruits from Red and Orange capsicum cultivars.

Plant Materials, Growth Conditions, and Fruit Collection
The experiment was conducted in a controlled environment greenhouse facility with east-west orientation located on the Hawkesbury Campus of Western Sydney University, Richmond, NSW, Australia. The four research bays (105 m 2 each) were fitted with HD1AR diffuse glass (70% haze, roof; 5% haze, wall), as previously described [13,59]. Two bays with diffuse glass were designated control bays and two bays (roof and side walls) were coated with SGF (ULR-80, Solar Gard, Saint-Gobain Performance Plastics, Sydney, Australia) and designated as treatment bays.
The experimental design involved a 2 × 2 factorial design testing two capsicum cultivars, Red and Orange (Gina and O06614, respectively, Syngenta, Durham, NC, USA). Seedlings were germinated for 42 days after sowing in Rockwool cubes (100 cm × 15 cm × 10 cm, Grodan, Roermond, The Netherlands) and transplanted (19 April and harvested 19 December 2019) into anchored Rockwool slabs contained within hydroponic gutters. Of the six gutters, the four middle gutters (10.8 m length × 0.25 m width, AIS Greenworks, Castle Hill, NSW, Australia) were alternatively planted with Red and Orange capsicum cultivars. The outer two gutters were planted with capsicum varieties, but not used for experimental data collection. Each slab supported four plants, such that each bay contained 40 experimental plant replicates. A trellis system was used to attain vertical plant growth. Plants were pruned to two main branches from the main stem that was allowed to grow 50 cm in height; weekly pruning was performed according to industry standards. Capsicum flowers were tagged upon evidence of pollination (August 2019) and fruits were harvested at 10-day intervals, starting 15 days after pollination (DAP) and ending at 65 DAP. A total of 12 samples from each cultivar per treatment at each fruit development stage were harvested, and the weight, diameter, length, and pericarp thickness were quantified.

Confocal Microscopy of Fruit Cuticle Thickness
Representative fruit samples were used to assess cuticle thickness using confocal microscopy, as previously described, with minor modifications [60]. Twelve different fruits from each genotype per treatment were harvested every 10 days. The exocarp cuticle from eight cross-sections of the equatorial region were quantified using an inverted laser scanning confocal microscope with a ×63 water immersion objective (Leica TCS SP5, Bensheim, Germany). Image processing was accomplished using LAS-AF 1.6.3 software. To avoid auto-fluorescence (cuticle, emission maximum = 475 nm), Nile red (Sigma Aldrich, St. Louis, MO, USA, 100 µg/mL methanol solution) and a cell wall-specific dye (Fluorescent bright 28; Calcofluor White M2R, Sigma Aldrich, St. Louis, MO, USA) dissolved in 0.01 M of distilled water were used to stain the samples for 10 min and 3 min, respectively. Excitation was set at 405 nm on the laser, and emission was collected between 420 and 436 nm (emission maximum at 428 nm) for the stained cell wall and between 597 and 649 nm (emission maximum at 620 nm) for the dyed cuticle. Two-dimensional and Z-stack images were obtained using a scan rate of 400 Hz in sequential scan mode to avoid interference between the two fluorophores. The step size was in the range of 1.0-1.5 µm and was determined via program auto-optimization, based on the input parameters of an individual Z-scan of the LAS-AF software (Leica Microsystems, Bensheim, Germany). The central region between pegs of the cuticle covering epidermal cells was used to estimate cuticle thickness and fruits were not affected by cuticle invaginations [32]. Image J (NIH, Bethesda, MD, USA) was used to determine the thickness of the cuticle (twelve biological replicates and eight technical replicates for each cultivar per treatment).

Postharvest Quality Trait Measurements
Fruit weighing at least 200 g were sampled 183 days after treatment (19 October 2019) and moisture content was determined [61]. Approximately 4 g of capsicum fruit equatorial pericarp was cut and oven dried at 70 • C for 48 h. Samples were cooled in a desiccator and weighed. This operation was repeated three times for each sample and expressed as a percentage. The pH of a liquid extract from 10 g of fruit sample (equatorial region) ground in 90 mL distilled water was determined [62]. Total soluble solids (TSS) were measured (three technical replicates per fruit and six biological replicates) using a digital refractometer (Mettler Toledo, Melbourne, Australia) using the liquid extract from the equatorial pericarp as previously described [62]. Ash was quantified using 3 g of the fruit equatorial zone and heated at 550 • C overnight into a grey-white ash as previously described [61]. Fruit colour was assessed upon harvest using a CR-400 chromameter (Minolta, Tokyo, Japan) calibrated against a standard white tile (Y = 84.8; x = 0.3199; y = 0.3377) prior to measurement [63]. Fruit surface colour measurements were taken at three points on the opposite sides to the equatorial region of the fruit. The Commission Internationale de l'Elcairage (CIE) L*, a*, b* colour scale was adopted, and the raw data was used to calculate chroma (C*) and hue angle (h) according to the formulas [C* = (a* 2 + b* 2 ) 0.5 ] and [h = arctan (b*/a*)] as described [64]. Ascorbic acid was quantified (g of ascorbic acid per kg fresh sample) by titration using 2, 6-dichloro-indophenol as previously described with minor modification [52]. Ten grams of samples was blended, and the pulp was homogenized with 50 mL of 12% oxalic acid. Titratable acidity (TA) was determined by titration using NaOH (0.1 N) as a standardized solution according to [62]. The juice from 10 g of fruit sample was added to 90 mL of distilled water and homogenized in a blender. Each replicate liquid was titrated three times to determine an average of titratable acidity (TA, g of citric acid mg/g of fresh sample).

Carotenoid Quantification
65 DAP fruit samples (12 replicates) were harvested for both control and SGF treatments, snap-frozen using liquid N 2 , and stored at −80 • C. Carotenoids were extracted under low-light conditions with 500 µL extraction buffer (60% ethyl acetate: 40% acetone and 0.1% butylated hydroxytoluene (BHT)) as previously described [65]. The liquid was partitioned into the ethyl acetate layer by adding 500 µL of H 2 O, centrifuged to separate the carotenoid-containing organic phase, and saponified to liberate free carotenoid molecules. Saponified carotenoids were analyzed by reverse-phase high performance liquid chromatography (HPLC, Agilent 1200 Series) using the GraceSmart-C30 (4 µm, 4.6 × 250 mm column; Alltech) column, and absolute total carotenoid levels (mg/kg of fresh weight) were calculated as previously described [66,67].

Quantification of Fruit Firmness, Water Loss, and Shelf Life
The harvested fruit firmness was determined using the XT plus texture analyzer (Stable Micro Systems, Godalming, UK) as described with minor modification using the Probe Volodkevich Bite Jaws (HDP/VB) [64]. A 3 cm diameter hole in the pericarp was punched in the equatorial region of the capsicum by a stainless steel punch [64]. The fruit skin was placed down on the steel plate and parameters established (pre-test speed 2 mm/s, test speed 3 mm/s, post-test 2 mm/s, compression deformation 30%, and trigger force 0.05 N). Water loss was determined using twelve ripened fruits (65 DAP) from each cultivar and treatment, and stored in 20 • C, 30% RH for 30 days. A high vacuum grease (silicone lubricant, Dow Corning) was applied to the surface of fruits to cover the pedicel scars. Water loss was calculated as the percentage of weight loss based on the initial fruit weight at 3, 5, 10, 15, 20, 25, and 30 days post-storage [68]. Capsicum shelf life was assessed by counting the days until visual shriveling and fruit firmness was evident as previously described [40]. A total of 36 fruits were harvested from each cultivar and treatment were stored in 2 • C/90% (temperature/relative humidity), 7 • C/90% and 20 • C/30% for 30 days. During storage, six fruits were photographed, and firmness measured after 0, 3, 5, and 15 days.

Statistical Analysis
All data and statistical analyses were performed under R 4.0.0 statistical computing environment. A p ≤ 0.05 is considered statistical significance. Levene's test from the car package analyzed the homogeneity of variance. Welch's t-test for unequal variances (with ≤0.05 probability for Levene's test) used the oneway.test function in R. The normal distribution was tested using the Shapiro-Wilk test for normal distribution, and statistically significant (≤0.05) parameters were then analysed using the Kruskal-Wallis test, which is a non-parametric equivalent of one-way ANOVA. Repeated measures ANOVA on different time point of measurements, such as fruit development, water loss, and firmness changes, used Mauchly's test (p ≤ 0.05), and pairwise comparison by the anova_test function from the rstatix package. Other packages were also used, including (but not limited to) Lubridate (for effective use of dates in plots), Sciplot (for plotting), and doBy (for calculating means and standard errors). The Pearson's correlation coefficient of PAR and quality parameters used the rcorr function of the Hmisc package. Principal component analysis (PCA) was used to test the multivariate associations among quality indexes, and the shelf-life-related parameters used the prcomp function.