Impact of Cold Stress on Physiological Responses and Fruit Quality of Shiranuhi Mandarin in Response to Cold Conditions

Abstract

We identified the minimum temperature limits to ensure Shiranuhi mandarin growth and fruit quality and provided overwintering temperature management guidelines. Expanded polystyrene panels with air conditioners were exposed to −1, −3, and −5 °C in the greenhouse for 15 h to determine the overwintering temperature. Leaves and fruits were analyzed at intervals for physiological response, fruit quality, and aromatic components. The low temperature treatment groups showed 1.3 to 1.4 times increased malondialdehyde content in leaves and 1.1 to 1.3-fold higher electrolyte linkage rates in the −5 °C treatment group alone. The sugar/acidity ratio was 1.1 to 1.3 times higher in the −5 °C treatment than in the control due to rapid acid reduction. The fruit firmness and citrus color index decreased notably after 21–28 days of treatment. Ascorbic acid content significantly decreased 17.3%–41.1% at −5 °C. Among the aromatic compounds, decanal levels notably increased with −5 °C treatment; −5 °C cold treatment notably affected oxidative stress in leaves and the sugar/acid ratio, ascorbic acid content, and aromatic compounds in fruits. If prolonged exposure to temperatures <−5 °C is expected, preharvest heating facilities are critical. We recommend maintaining greenhouse temperatures >0 °C during fruit growth and >−3 °C after harvest as the minimum temperature to preserve fruit set and quality.

1. Introduction

Citrus (Rutaceae), a genus of flower- and fruit-producing trees and shrubs, is grown worldwide in tropical and subtropical climates. The tree’s vigor, fruit quality, and yield vary substantially depending on the climate. The major growing regions are between 40° north-south latitudes and extend along the equator. Citrus fruits are sensitive to cold temperatures [1,2]. The cold tolerance of plants, specifically a citrus variety, can vary in degree based on factors such as the variety itself, its cold hardening status, and the specific tissue being considered [2,3,4].

Jeju Island, the main citrus-producing area in Korea, has a subtropical climate that allows cultivation of horticultural crops in winter. Global warming is increasingly impacting ecosystems, and several researchers have predicted that extreme rainfall or temperature events will become more frequent in the future, leading to an increase in weather disasters such as cold waves on the Korean Peninsula and Jeju Island [5,6,7]. In fact, anomalous cold waves have occurred frequently in Jeju in recent years [8]. Shiranuhi mandarins [(Citrus unshiu × C. sinensis) × C. reticulata)] are sweet, large, easily peelable fruits that are favored by domestic consumers, making them the second highest yielding variety following the Satsuma mandarin (C. unshiu Marc.) [9,10]. Due to global warming and expectations as a new income crop, its cultivation area has recently expanded to mainland areas in Korea, including the southern coast [11]. However, its fruiting season is longer than that of C. unshiu, and it is harvested from January to March of the following year, making it vulnerable to increasing cold wave damage. Cold stress has a negative impact on fruit quality, which is determined by various factors including sweetness, flavor, firmness, and color of peel and flesh [1]; hence, it is crucial to analyze the temperature at which physiological changes in the tree and fruit begin to occur and establish a low-temperature threshold to prepare for the damage caused by this sudden cold.

Tropical and subtropical crops are particularly prone to chilling injuries (CIs) when temperatures fall below critical levels [12]. Many studies have been conducted on the physiological responses of those crops, such as molecular responses due to chilling injury, physiological responses related to photosynthesis, and hormonal changes. CI can reduce leaf expansion, wilting, yellowing, and tissue necrosis, whereas freezing stress causes severe membrane damage due to acute dehydration. Furthermore, stress-induced reactive oxygen species (ROS) can damage membranes [13]. In this study, we analyzed malondialdehyde (MDA) and electrolyte linkage (EL) to investigate the degree of stress in the leaves and fruits of Shiranuhi mandarins at various freezing temperatures. MDA is a final product of lipid peroxidation and is commonly used as a biological indicator of oxidative stress. It can be easily quantified spectrophotometrically using a thiobarbituric acid-reacting substance (TBARS) assay [14,15,16]. Additionally, EL has been widely used to determine the degree of stress tolerance in plant cells by measuring electrical conductivity [17,18,19]. Prolonged exposure of tomatoes to low temperatures causes freezing damage, negatively affecting the composition of volatile components and flavors [20,21]. Citruses, including orange and Shiranuhi mandarin, have also been reported to be affected by changes in specific aromatic components under frost conditions [22,23,24]. Though many researchers have worked on the physiological responses of citrus trees, very few researchers have reported on changes in fruit quality after chilling injuries.

The purpose of this study was to provide guidance on winter temperature management for Shiranuhi mandarin, a freezing stress-susceptible citrus cultivar. In this study, we investigated cold stress responses of Shiranuhi mandarins, and their effects on fruit quality were investigated by analyzing total soluble solids (TSS), titratable acidity (TA), firmness, free sugars, organic acids, ascorbic acid (vitamin C), and aromatic components.

2. Materials and Methods

2.1. Plant Materials and Experimental Freezing Treatments

The study was carried out in mid-January 2022 at the Citrus Research Institute of the Rural Development Administration (RDA), Seogwipo-si, Jeju-do, Korea (N 33°18′06.0″, E 126°36′39.6″), and applied to 250 days after full bloom on 12-year-old bearing trees growing in a greenhouse. Trees with similar growth and fruiting statuses were selected to serve as treatments. The freezing treatment was modified using the methods described by Kim et al. [22]. For each treatment, an individual cage was assembled using 10 cm-thick expanded polystyrene panels. These panels had dimensions of 2 m in diameter and 2 m in length, effectively enclosing the trees on five sides. The freezing treatments lasted about 15 h at temperatures of −1, −3, and −5 °C, respectively, from 5 p.m. to 8 a.m. the next day using an outdoor air conditioner unit. This duration of treatment is similar to the 15 h of temperatures below −5.5 °C experienced during the record cold wave on Jeju Island in 2016. Control trees were maintained at a temperature of 1.2 °C. The temperature was monitored using a thermocouple data logger (Hobo UX120-014M; Onset Computer Corp., Bourne, MA, USA). Distinct cold treatments were assigned, with temperature variations of ±0.6 °C. The average day/night air temperatures in the greenhouse during the 7 to 28 days after treatment were 9.7 °C and 2.6 °C, respectively.

2.2. Lipid Peroxidation Analysis

The malondialdehyde (MDA) content for the analysis of lipid peroxidation was measured using the method described by Jakhar and Mukherjee et al. [16]. MDA content was determined using a UV spectrophotometer (UV-2700; Shimadzu Corp., Seoul, Korea). The fruit in the middle of the tree, and the 3rd and 4th leaves from the one-year-old shoots at 7 and 28 days after both the freezing temperature and non-treatment, were randomly collected. Six or nine replicates were assayed per treatment. The fresh leaves, fruit peels, and pulps collected were immediately immersed in liquid nitrogen after sampling. Fresh samples (200 mg) were ground into a powder using liquid nitrogen, homogenized with 2 mL of 50 mM phosphate buffer (pH 7.0), and centrifuged at 3500 rpm for 15 min at 4 °C using a centrifuge 5810R (Eppendorf). A 1 mL supernatant was taken in a new tube and added to 2 mL of 0.5% thiobarbituric acid in 20% trichloroacetic acid. The mixture was incubated in a water bath for 30 min at 95 °C and then rapidly cooled in an ice bath. The samples were centrifuged at 3500 rpm for 10 min at 4 °C. The supernatants’ optical densities were recorded at 532 and 600 nm. All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). The MDA content was calculated using an extinction coefficient of 155 mM⁻¹ cm⁻¹.

2.3. Electrolyte Leakage Analyses

The leaves were collected from one-year-old shoots at 7, 14, 21, and 28 days after freezing temperature treatment and non-treatment. Three replicates were assayed per treatment at 7, 14, 21, and 28 days after freezing temperature treatment and non-treatment. Nine replicates were assayed per treatment. EL analysis was conducted using the method described by Kim et al. [17]. Briefly, the disks were incubated in 10 mL of distilled water for 2 h at 32 °C, and the first electrical conductance (EC₁) was measured using a CHS30 EC meter (Mettler Toledo Inc., Greifensee, Switzerland). The disks were then autoclaved at 121 °C for 20 min, then cooled, and then the second conductance (EC₂) was measured. The EL was calculated as follows:

EL(%) = EC₁/EC₂ × 100

2.4. Fruit Properties Analyses

Fifteen fruits were randomly collected 7, 14, 21, and 28 days after freezing treatment and non-treatment to evaluate the effect on fruit quality characteristics. Hunter’s L, a, and b color scales were measured using a CR-400 Chroma Meter (Konica Minolta Sensing Inc., Osaka, Japan), and the values were averaged for three random equatorial belts on the fruit surface. Calibration was performed using a white calibration plate provided by the manufacturer. The citrus color index (CCI) was calculated using Hunter’s L, a, and b values according to the method described by Jimenez-Cuesta et al. [25]. Firmness was assessed using a 3-mm probe attached to a texture analyzer (TA-XT2; Stable Microsystem Ltd., Surrey, UK). After removing the rind and squeezing the juice, the TSS content was measured using a refractometer (PAL-1, Atago Co. Ltd., Tokyo, Japan). To analyze the TA, each juice sample was diluted five times with distilled water and titrated using a 0.1 N sodium hydroxide solution with a 1% phenolphthalein solution. The collected juice sample was immediately stored at −80 °C and used for free sugar and organic acid analyses.

2.5. Free Sugars, Organic Acids, and Ascorbic Acid Determination

After the TSS and TA analyses, juice was used for free sugar and organic acid analysis. Six replicates were assayed per treatment. To measure free sugars and organic acids, the juice was diluted 10-fold and 5-fold with distilled water and filtered using a 0.2-μm filter. Free sugars, organic acids, and ascorbic acid were quantified using a Shimadzu Prominence UFLC system (Shimadzu Co., Ltd., Kyoto, Japan) equipped with an RID detector (RID −20A) and a UV-visible detector (SPD 20A). Columns and mobile phases were prepared as described by Kim et al. [22]. HPLC-grade standards were used to quantify sucrose, fructose, glucose, citric acid, malic acid, and ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA).

2.6. Volatile Compound Analysis Using GC-MS

After the TSS and TA analyses, the juice was used for volatile compound analysis, and three replicates were assayed per treatment. The volatile compounds were identified by headspace-solid-phase microextraction (SPME) using a gas chromatograph 7890A GC system (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a 5975C Inert XL MSD (Agilent Technologies Inc., Santa Clara, CA, USA). Thirty milliliters of juice samples were placed in a glass vial at 60 °C on a stirring hot plate for 15 min, allowing for volatile organic compounds to be adsorbed into SPME fiber (Supelco, Bellefonte, PA, USA) and then desorbed for 1 min. The non-polar DB-5ms column (0.25 μL × 30 m × 0.25 mm; Agilent Technologies Inc., Santa Clara, CA, USA) was used. The GC oven temperature was programed from 40 °C (for 3 min) to 90 °C (4 °C/min) to 210 °C (19℃/min) and finally to 210 °C (3 min). The temperature of both the inlet and detector was 230 °C, and the flow rate of helium as the carrier gas was 2.2 mL/min. Data processing for volatile compounds was performed using Agilent MSD ChemStation E.0.201.1177 software.

2.7. Statistical Analysis

Analysis of variance (ANOVA) was applied using R version 3.6.3 of the R software package (R Studio, Boston, MA, USA). The mean values and their standard deviation were separated using Scheffe’s post-hoc test at p < 0.05 for treatment comparisons. Pearson’s correlations between the different parameters were calculated using SPSS 21 (SPSS Inc., Chicago, IL, USA).

3. Results

3.1. Plant Response to Cold Treatment in Relation to Lipid Peroxidation

In both the control and low-temperature treatment groups, the MDA content, which is an indicator of the degree of oxidative stress caused by lipid peroxidation, was highest at 7 and 28 days after cold treatment (15 h) in the order of leaf, peel, and pulp (Figure 1). The MDA content in the leaves of the cold-treated groups ranged from 123.5 to 144.1 nmol/g at 7 and 28 days after treatment. This was 1.21 to 1.36 times higher than the control group, showing 98.3 to 105.5 nmol/g. However, there was no significant difference in the MDA content between the fruit peel and pulp.

This study measured electrolyte leakage from the leaves at 7, 14, 21, and 28 days after treatment. The −5 °C treatment showed considerably higher leakage (13–18%) than the other treatments (Figure 2).

3.2. Changes in Fruit Quality

The fruit rind that has been severely damaged by freezing becomes damp due to leaked moisture, and when the fruit is split in half, the walls of the segments are wet. Figure 3 illustrates the appearance of the inner part of the fruit after 28 days of low-temperature treatment. While the walls of the control group, the −1 °C, and the −3 °C treatment groups were unaffected, the −5 °C treatment group exhibited wet segment walls and signs of damage, such as discoloration and shrinkage (Figure 3). A detailed investigation conducted on the 28th day after treatment further supported these findings, with approximately 75% of the samples in the −5 °C treatment group showing wet segment walls and 45% of the samples displaying signs of off-flavor (Table S1).

Fruit quality analysis was conducted based on the number of days after treatment and the cold treatment temperature (Figure 4). The control group showed a total soluble solid content of 12.5°Brix and 13.0°Brix at 7 and 14 days after treatment, respectively, significantly higher than the other cold treatment groups (Figure 4A). However, 21 days after treatment, the control and the −5 °C treatment groups exhibited considerably higher total soluble solid content than the other treatment groups. By the 28th day, the −5 °C treatment group displayed the highest total soluble solid content. The acid content ranged from approximately 1.19% to –1.26% in the control group, which was significantly higher than the other cold treatment groups regardless of the number of days after the treatment (Figure 4B). In the −1 °C and −3 °C treatment groups, the acid content decreased 21 days after treatment, while in the −5 °C treatment group, it consistently decreased from 7 to 28 days after treatment, reaching 0.98%. While the TSS/TA ratio of the control treatment increased slightly with the number of days after treatment, the TSS/TA ratio of the −5 °C treatment increased significantly from 11.1 to 13.1, which is 1.1 to 1.3 times that of the control (Figure 4C). Seven days after treatment, the hardness of the fruit in the −5 °C group was 12.8 N. However, 21 and 28 days after treatment, the hardness values of all cold treatment groups indicated 10.0–10.1 N, representing a 20.6% to 29.9% reduction compared to the control group’s hardness values of 13.2 to 14.3 N (Figure 4D). There was no notable difference in CCI between the treatments after 7 and 14 days. However, after 21 days of treatment, the CCI value in the low-temperature treatment group was lower than that in the control group (Figure 4E).

3.3. Changes in Free Sugars, Organic Acids, and Ascorbic Acid

Table 1. Impact of interaction between freezing treatment temperature and period after treatment on free sugar contents in Shiranuhi mandarin (mg/mL fresh juice).

Days after Treatment (DAT)	Temperature Treatments	Fructose	Glucose	Sucrose	Total
7	control	24.5 ± 2.0 a	26.6 ± 3.1 a	60.5 ± 8.0 a	111.6 ± 11.0 a
	−1 °C	22.0 ± 2.2 ab	24.3 ± 3.8 a	61.7 ± 4.5 a	107.9 ± 8.7 a
	−3 °C	23.5 ± 2.2 ab	26.1 ± 3.7 a	60.0 ± 3.3 a	109.6 ± 8.1 a
	−5 °C	21.4 ± 0.9 b	23.4 ± 2.7 a	56.6 ± 2.2 a	101.3 ± 4.2 a
14	control	23.9 ± 2.5 a	25.4 ± 2.6 ab	64.5 ± 3.2 a	113.8 ± 7.3 a
	−1 °C	20.7 ± 1.1 a	22.8 ± 1.0 b	57.2 ± 4.5 b	101.0 ± 5.1 b
	−3 °C	24.2 ± 2.3 a	28.2 ± 3.1 a	59.6 ± 2.1 ab	112.0 ± 5.3 ab
	−5 °C	22.4 ± 2.1 a	24.9 ± 3.3 ab	59.8 ± 3.7 ab	107.0 ± 8.0 ab
21	control	20.1 ± 1.0 b	19.4 ± 1.6 c	66.8 ± 1.7 a	106.3 ± 3.2 ab
	−1 °C	21.1 ± 2.3 b	21.0 ± 2.4 bc	58.2 ± 4.2 b	100.3 ± 6.6 b
	−3 °C	24.6 ± 1.9 a	25.0 ± 2.3 a	59.4 ± 2.1 b	109.1 ± 4.3 ab
	−5 °C	23.5 ± 2.1 ab	23.4 ± 2.7 ab	62.7 ± 2.6 ab	109.6 ± 6.3 a
28	control	20.4 ± 1.8 bc	23.9 ± 3.7 bc	65.3 ± 3.8 a	109.7 ± 6.1 ab
	−1 °C	19.7 ± 0.9 c	23.2 ± 1.0 c	61.8 ± 4.3 a	104.7 ± 5.4 b
	−3 °C	22.2 ± 2.2 ab	28.2 ± 3.9 a	56.1 ± 4.7 b	106.6 ± 8.9 ab
	−5 °C	22.9 ± 1.2 a	28.0 ± 3.4 ab	62.6 ± 2.4 a	113.5 ± 5.6 a

Values (mean ± SD) denoted by the same letter superscript within a column on each date are not significantly different (p < 0.05, Scheffe’s test), following a two-way ANOVA with temperature and days after treatment as factors of variability.

presents the fructose, glucose, and sucrose levels based on the days after treatment and the cold treatment temperature. Fructose levels were the lowest at 21.4 mg/mL at −5 °C on day 7 after treatment and substantially higher than other treatment groups, reaching 22.9 mg/mL on day 28 after treatment in the −5 °C treatment group. Glucose levels ranged from approximately 23.4 to 26.6 mg/mL on day 7 after treatment, showing no difference between treatments. However, in the −3 °C group, glucose levels increased to approximately 25.0–28.2 mg/mL after 14 days, notably higher than the other treatments. On the 7th day after treatment, there was no significant difference in the sucrose content between the treatments, but the control group exhibited the highest levels at 64.5 and 66.8 mg/mL on the 14th and 21st days after treatment, respectively. In contrast, on the 28th day after treatment, the −3 °C treatment group showed the lowest sucrose content at 56.1 mg/mL compared to other treatment groups, including those at higher temperatures. Similarly, total free sugar content did not differ significantly between treatments 7 days after treatment, ranging from approximately 101.3–111.6 mg/mL. However, 14 days after treatment, the control group exhibited higher levels of total free sugar content at 113.8 mg/mL than the low-temperature treatment group. On the 21st and 28th days after treatment, the −5 °C group had considerably higher levels of total free sugar content than the other treatment groups.

Table 2. Impact of interaction between freezing treatment temperature and period after treatment on contents of organic acid and ascorbic acid in Shiranuhi mandarin.

Days after Treatment	Temperature Treatments	Organic Acids (mg/mL Fresh Juice)			Ascorbic Acid (mg/100 mL Fresh Juice)
		Malic Acid	Citric Acid	Total
7	Control	2.5 ± 0.4 a	11.4 ± 0.9 ab	13.9 ± 1.0 ab	43.2 ± 4.0 a
	−1 °C	2.7 ± 0.2 a	10.7 ± 1.2 ab	13.4 ± 1.1 ab	41.1 ± 3.0 ab
	−3 °C	2.6 ± 0.2 a	11.7 ± 1.2 a	14.3 ± 1.1 a	43.7 ± 6.5 a
	−5 °C	2.6 ± 0.2 a	10.0 ± 0.9 b	12.6 ± 0.8 b	35.7 ± 6.2 b
14	control	2.6 ± 0.3 a	11.8 ± 0.6 a	14.4 ± 0.7 a	44.0 ± 2.6 a
	−1 °C	2.5 ± 0.3 a	11.3 ± 1.0 ab	13.8 ± 0.9 ab	42.3 ± 4.7 a
	−3 °C	2.4 ± 0.1 a	11.1 ± 0.9 ab	13.5 ± 0.9 ab	39.7 ± 2.9 a
	−5 °C	2.3 ± 0.1 a	10.2 ± 1.0 b	12.5 ± 1.0 b	32.1 ± 4.4 b
21	control	2.3 ± 0.2 ab	12.8 ± 1.0 a	15.1 ± 1.0 a	44.1 ±4.1 a
	−1 °C	2.5 ± 0.2 a	10.5 ± 0.7 b	13.0 ± 0.5 b	43.0 ±5.3 a
	−3 °C	2.4 ± 0.2 a	10.3 ± 0.6 b	12.7 ± 0.6 b	41.6 ± 4.8 a
	−5 °C	2.0 ± 0.1 b	9.8 ± 1.1 b	11.9 ± 1.1 b	26.0 ± 5.0 b
28	control	2.5 ± 0.4 a	11.2 ± 1.5 a	13.6 ± 1.3 a	46.0 ± 3.3 a
	−1 °C	2.4 ± 0.1 a	9.2 ± 0.5 b	11.6 ± 0.5 b	42.8 ± 3.1 a
	−3 °C	2.3 ± 0.1 a	9.8 ± 0.7 b	12.1 ± 0.7 b	43.6 ± 5.2 a
	−5 °C	1.8 ± 0.2 b	9.4 ± 0.9 b	11.2 ± 0.8 b	35.1 ± 3.7 b

Values (mean ± SD) denoted by the same letter superscript within a column on each date are not significantly different (p < 0.05, Scheffe’s test), following a two-way ANOVA with temperature and days after treatment as factors of variability.

displays the organic acid content based on the days after treatment and the low-temperature treatment temperatures. The malic acid content remained relatively stable, ranging between 2.3 and 2.7 mg/mL up to 14 days after treatment, with no significant difference observed among the treatment groups. However, after 21 days, malic acid content significantly decreased in the −5 °C treatment group. The citric acid content was also significantly lower, measuring 10.0 mg/mL and 10.2 mg/mL, in the −5 °C treatment group at 7 and 14 days after treatment. After 21 days, the citric acid content showed a significant decrease in all treatment groups compared to the control group. Overall, the total organic acid content was the lowest in the −5 °C treatment group at 7 and 14 days after treatment, measuring 12.5 mg/mL and 12.6 mg/mL, respectively, with citric acid accounting for over 80% of this reduction. Additionally, the total organic acid content significantly decreased in all cold-treated groups after 21 days of treatment.

The ascorbic acid content of the control group ranged from 43.2 to 46.0 mg/100 mL, while in the −5 °C treatment group, it significantly decreased by 17.3 to 41.1% compared to the control group, ranging from 26.0 to 35.7 mg/100 mL, depending on the days after treatment.

3.4. Changes in Volatile Compounds

In the present study, analysis of the aromatic compounds following cold treatment revealed the identification of 15–32 compounds, which accounted for 97.3–99.5% of the total aromatic compounds (Figure 5). Monoterpene hydrocarbons comprised approximately 78.6–93.3% of the identified compounds, while two monoterpene alcohols contributed around 2.5–11.2%. The remaining compounds exhibited diverse combinations based on the days after treatment. Notably, limonene, the predominant aromatic compound, ranged from 68.55% to 82.29%, depending on the number of days after treatment in the control group. The highest number of compounds was observed 21 days after treatment with octanal, nonanal, decanal, and perilla aldehydes, which were considerably higher at −5 °C Decanal, in particular, demonstrated a substantial increase at −5 °C regardless of the number of days after treatment (Table S2).

3.5. Correlation between Fruit Quality Parameters

Pearson correlation coefficients were analyzed to evaluate the correlation between parameters associated with fruit quality according to the temperature treatment used and are summarized in

Table 3. Pearson’s correlation analysis among fruit quality parameters of the Shiranuhi mandarin fruit either after exposure to normal or freezing temperature treatments with the day after treatment.

Quality Parameters	TSS	Acidity	FN	CCI	TFS	RSU	NRSU	MA	CC	TOA
Acidity	0.227 ***
FN	0.056	0.261 ***
CCI	0.174 **	−0.63	0.406 ***
TFS	−0.109	−0.083	−0.096	0.064
RSU	−0.028	−0.217 *	−0.019	0.068	0.737 ***
NRSU	0.185 *	0.083	−0.124	0.028	0.772 ***	0.139
MA	0.023	0.058	−0.180 *	−0.418 ***	−0.047	−0.143	0.065
CC	0.003	0.044	−0.065	−0.256 **	0.086	−0.003	0.128	0.092
TOA	0.014	0.055	−0.102	−0.336 ***	0.075	−0.033	0.141	0.317 ***	0.973 ***
AA	0.020	−0.140	−0.133	−0.179 *	0.148	0.075	0.146	0.272 **	0.476 ***	0.512 ***

*, **, *** Correlation is significant at 0.05, 0.01, and 0.001, respectively. FN = Firmness, CCI = Citrus color index, TFS = Total free sugars, RSU = Reducing sugars, NRSU = Non-reducing sugar, MA = Malic acid, CC = Citric acid, TOA = Total organic acid, AA = Ascorbic acid.

. Sugar content was significantly positively correlated with acidity, CCI value, and non-reducing sugars (Frucotse+Glucose). Acid content showed a significant positive correlation with hardness and a significant negative correlation with reducing sugars (Sucrose). Hardness was significantly positively correlated with CCI. Furthermore, CCI was significantly negatively correlated with malic acid. Non-reducing sugars were positively correlated with Sugar Content and Total Free Sugars. Citric acid was significantly negatively correlated with CCI. Total organic acids were significantly inversely correlated with CCI, with significant correlations for malic and citric acids. Vitamin C was significantly positively correlated with malic and citric acids and total organic acids, and negatively correlated with CCI. Of these, the highest definitional correlations were 0.737 (p < 0.001) for total free sugars and reducing sugars, followed by 0.772 (p < 0.001) for total free sugars and non-reducing sugars, 0.973 (p < 0.001) for citric acid and total organic acid, and 0.512 (p < 0.001) for vitamin C and total organic acid. These results suggest that sugar content, acid content, CCI, non-reducing sugars, citric acid, and vitamin C should be considered important factors for maintaining fruit quality in winter.

4. Discussion

The MDA content in the leaves significantly increased after the low-temperature treatment compared to the control, while there was no difference in the fruit, including peel and pup. This result indicates that Shiranuhi mandarin leaves are more sensitive to freezing than fruit. Previous studies reported that MDA content increases in response to various stresses: in pepper plants subjected to salinity stress [26], in sun-damaged mandarin fruits [27], and cold-sensitive blueberry fruits stored at 0 °C for 30 days after harvest [28]. Morales and Munné-Bosch reported that while a sustained increase in MDA levels resulting from external stress could lead to the inactivation of protein structure, photosynthesis-related proteins, and photosystems, ultimately causing a loss of membrane fluidity, a temporary increase in MDA can activate regulatory genes associated with plant defense and development, thereby protecting cells from oxidative stress conditions [29]. The cold resistance of tangerine fruit is weaker than that of leaves [2], but it is considered that the treatment temperature and duration of low temperature in this experiment was not at a level that could affect lipid peroxidation accumulation in fruit.

Electrolyte leakage is a plant’s stress response and is primarily associated with the loss of potassium ions, which are abundant in plant cells. Stress-induced leakage is often accompanied by the generation of ROS and can lead to programmed cell death [18]. In our previous study with Shiranuhi mandarin trees, a −7 °C treatment for 6 h induced severe damage to the leaves and branches, while a −5 °C treatment caused relatively weak damage, and the critical temperature (LT50) of Shiranuhi mandarin in vivo was reported to be about −6.5 °C [17,22]. It is difficult to compare the results of the previous study with those of the current study since the freezing treatment time is different. However, the leaves may be damaged at −5 °C or lower, while over −3 °C for 15 h of low-temperature treatment, there is no significant difference from the control in electrolyte leakage, so it seems that cell necrosis does not occur.

Typically, citrus fruits exhibit an increase in the total soluble solid and a decrease in acidity content as they mature, and the acid content decreases with the color parameters increasing from the early stages of coloring to the harvest season. However, it noted that frozen-damaged fruits exhibited low TSS, acid content, and poor fruit quality [1]. This is because the formation of ice crystals resulting from freeze injury damages the juice vesicles in the citrus segments, causing the juice to leak out of the fruit and resulting in dehydration [30]. Increased TSS/TA of the fruit is positive for quality, but water content increases after 14 days of treatment below 1 °C (Figure S1), which can eventually lead to juice sac drying. The °Brix measurement at −5 °C treatments appears to be elevated due to water leakage, as it can be affected by dissolved substances other than sugars. In a prior study by Kim et al., exposure of Shiranuhi mandarin fruits to −5 °C for 6 h did not result in a significant difference in fruit quality compared to the unfrozen control group [22]. However, treatment at −7 °C significantly decreased total soluble solid content, color difference, and hardness, while acidity remained unchanged. In our study, the rapid decrease in acidity observed in the −5 °C treatment is likely attributed to the longer treatment time at that temperature.

Our study found that the −1 and −3 °C treatment groups had the lowest free sugar content compared to the other groups after 28 days, which may have important implications for future research in this field. Notably, Kim et al. observed a significant decrease in sucrose levels 14 days after treatment in Shiranuhi mandarin trees subjected to low-temperature treatment at −7 °C for 6 h [22]. Sugar is a primary metabolite synthesized in the leaves and delivered to the fruit. It provides sweetness and serves as a signal for environmental stimuli [31]. Most intracellular sugars are located in vacuoles. Researchers have found that under optimal conditions with high sucrose availability in fruits, invertase and sucrose synthase produce sufficient amounts of hexoses (glucose) to activate cell division, promote the production of non-enzymatic antioxidants, and prevent programmed cell death (PCD). However, under stressful conditions, the effectiveness of sucrose is limited; invertase and sucrose synthase activities are inhibited, leading to PCD and the cessation of cell division. In this study, a temperature below −1 °C affected the composition of sucrose, fructose, and glucose, which is considered a stress condition for Shiranuhi mandarin fruits.

Total organic acid content was lower in all cold treatments compared to the control 21 days after cold treatment, with the −5 °C treatment showing a significant decrease. Consistent with our findings, a previous study by Kim et al. reported a significant reduction in citric acid, malic acid, and total organic acid content 14 days after treatment on Shiranuhi mandarin fruits kept below −5 °C for 6 h [22].

Citrus is a significant dietary source of ascorbic acid (vitamin C), and its content is influenced by various factors, such as citrus varieties, production factors, climatic conditions, and maturity [1,32]. In our study, the ascorbic acid content in the −5 °C treatment group showed a significant reduction of 17.3 to 41.1% compared to the control group, regardless of the number of days after treatment. Previous research has highlighted that enzymes such as cytochrome oxidase, ascorbic acid oxidase, and peroxidase can destroy ascorbic acid in citrus, and the loss of ascorbic acid tends to increase with juice storage time and temperature [32]. Additionally, the susceptibility of citrus to cold-induced disorders, such as CI during cold storage, has been found to correlate positively with ascorbic acid content [33]. Studies have also linked the loss of ascorbic acid in orange juice to the oxygen permeability of packaging materials [34]. Furthermore, ascorbic acid degradation in refrigerated pineapples occurs even before visible symptoms of deterioration [35]. Based on these findings, it can be inferred that low temperatures at −5 °C may contribute to the destruction of ascorbic acid in citrus juice, leading to diminished fruit quality in Shiranuhi mandarin.

Terpenes, sulfur compounds, amino acids, carbohydrates, carotenoids, vitamins (B1, C), phenolic acids (ferulic acid), and other aromatic compounds have been found to decompose during citrus processing and storage. Off-flavors in juices are believed to be caused by the formation of specific compounds, including terpene alcohols, hydrocarbons, oxides, volatile sulfur compounds, furaneol, citral, and 2-methyl-3-furanthiol [36]. In our study, the rate of off-flavor was significantly high in the −5 °C treatment group, accounting for 45%, and the decanal levels considerably increased, regardless of the number of days treated. Previous studies have reported changes in the composition of monoterpenes, sesquiterpenes, and other compounds in response to cold treatment [22], but no specific changes in decanal levels were observed. Decanal, a non-terpenoid aldehyde, is known for its various flavor notes, depending on the citrus variety and extraction site, such as oily, fatty, bergamot-like, herbal, beefy, and lemon/citrus [37,38,39]. The association between off-flavor incidence and decanal may be worthy of further study. Further, since the low-temperature treatment time was limited, it is necessary to study the treatment time and try various methods to replace heating.

5. Conclusions

Extreme weather events, particularly cold surges due to climate change, are causing growing concern about the potential negative impacts on citrus tree growth and fruit quality. This study demonstrated that Shiranuhi mandarins experience physiological changes and CI under cold conditions, impacting fruit quality parameters, such as TSS, acidity, color, and flavor. To preserve citrus physiology and fruit quality during sudden cold waves, it is essential to maintain an internal greenhouse temperature above 0 °C when the fruit is hanging and above −3 °C when the fruit is not hanging. Understanding these critical temperatures can help farmers proactively implement appropriate protective measures, such as managing the temperature inside the greenhouse during a sudden cold wave and utilizing protective coverings to mitigate chilling. In addition, understanding the relationship between physiological changes and fruit characteristics may provide a basis for further research on storage or transportation to maintain fruit quality during winter distribution.