3.1. Effects of Excessively High Temperatures on Shoot Growth and Fruit Quality
In citruses, under the high temperatures prevailing in the tropics, citrus fruit development is fast, and the fruits become very large. The heat unit requirements for the maturation of Valencia oranges grown in cool regions is twice that of Valencia oranges grown in tropical regions [
11]. In the present study, an increase in the daytime and/or night-time temperature also promoted increases in the transverse and longitudinal diameters of the fruits, and a large difference in longitudinal diameter was observed (
Figure 2b). Moon et al. [
12] similarly reported vigorous enlargement of Shiranuhi mandarin fruits through spring heating treatment compared to a control treatment. The author also observed that the increase in longitudinal diameter was more vigorous than that of the transverse diameter. Therefore, it seems that high temperature at the fruit development stage is closely related to fruit enlargement.
In common mandarins such as those of C. unshiu, the neck at the fruit stem end reduces market value because of its poor appearance. However, this is an inherent characteristic of the cultivar Shiranuhi, and it acts as an important factor in determining market value. There was a strong correlation between the longitudinal diameter of the fruit and the length of the neck of the fruit (0.67, p < 0.01; data not shown). High temperature also seems to be a factor that promotes neck development at the fruit stem. Excessively high temperatures during the day seemingly have a greater effect on the neck of the fruit stem. Moreover, excessively high temperatures during the day cause a high daily temperature range, which appears to have a greater influence on the neck of the fruit stem.
High-temperature treatments in plastic houses resulted in pummelo (
C. grandis L. Osbeck) fruits with pyriform shapes and prominent necks at the stem end, and these fruits had the shortest transverse/longitudinal diameters [
13]. In the present study, the number of shoots and shoot length under the D8 treatment were greater than those under the other treatments. This was the result of an increase in vegetative growth (fresh weight) as the fruit set decreased (
Table 1). On the other hand, the N4 treatment presented the lowest number of shoots; however, the fruit set was higher than those under the other treatments, and the yield was lower than those under the Con and D4 treatments. The most significant factors for heat stress-related yield loss for cereals include a shortened developmental phase, reduced light perception, and changes in the processes associated with transpiration, photosynthesis, and respiration [
5,
14].
Continuous fruit decay occurred in the trees after the colouration period, and the D8 treatment resulted in 2.5-fold more continuous fruit decay than the Con treatment did (data not shown). This difference is thought to have been caused by the generation of dew on the pericarp caused by heat from the ground. The TSS/total acidity ratio is a physiological parameter that can determine fruit quality. The standard for Shiranuhi mandarin fruit quality in Korea is greater than 12° Brix for the TSS content and 1.1% acidity. In our study, a markedly increased daytime temperature (D8) reduced the accumulation of sugars, while an increased daytime/night-time temperature decreased the acidity. In particular, days and nights with increased temperatures seemingly had a greater effect on acid accumulation than days or nights with normal temperature alone. Previous studies have shown reduced TSS contents and citric acid contents in citrus fruits under high-temperature treatment [
12,
13]. Hutton and Landsberg [
15] predicted a decrease in TSS and acidity with a rise in temperature sums over time (effective heat units) during orange fruit development. In kiwifruit, heating during the starch accumulation period significantly reduced the contents of carbohydrates, while heating during the fruit maturation period reduced the starch content and delayed fruit maturity, showing different trends according to heating duration [
16]. Grigenberger et al. [
17] proposed that elevated temperature leads to increased rates of respiration, resulting in a decline in 3-phosphoglyceric acid, which then inhibits enzyme and starch synthesis in potato tubers, indicating reduced starch contents. Moon et al. [
12] also reported that the photosynthesis rate, stomatal conductance, and transpiration rate of citrus leaves during a spring heating treatment were much higher than those under a control treatment. Stress treatments, such as high temperature, drought, and high temperature/drought stress, caused reduced sucrose and starch contents in wheat grain [
18]. The authors found that with the stress treatments, key enzyme activities and the related gene expression led to not only repressed conversion of sucrose to starch, but also a decrease in sucrose content. Therefore, it is thought that the increase in respiration rate due to high temperatures interferes with the transfer of photo-assimilates from leaves to fruits, and analysis of enzymes associated with sugar or acid synthesis or related gene-level studies are needed to confirm this phenomenon.
Pericarp colour is another important factor that determines fruit quality. The pigmentation of mandarin and oranges varies greatly among species, and the two main types of isoprenoid-derived pigments (chlorophylls and carotenoids) are responsible for citrus fruit colouration [
19]. When a citrus fruit matures, the amount of chlorophyll decreases, and carotenoids accumulate. Spiegel-Roy and Goldschmidt [
11] reported that the decrease in chlorophyll in citrus rinds coincides almost with the onset of carotenoid accumulation. With respect to the colour of Shiranuhi mandarin pericarps in this study, the difference between the
L and
b values decreased considerably at harvest time, but the
a value clearly differed between the treatments (
Figure 3b). The increased temperature during the day seemed to have a negative effect on fruit colouration. Itle and Kabelka [
20] suggested that the
L* colour value correlated negatively with lutein and total carotenoid contents, whereas the
a* and
b* colour values were strongly correlated with total carotenoids and lutein, respectively. The authors suggested that a negative correlation between
L* and certain carotenoids would be expected, because any increase in pigment content would increase darkness and thereby decrease the
L*. Matsumto [
21] also reported that the expression of carotenoid biosynthesis-related genes in citrus fruit are sensitive to temperature. In subtropical areas, pigmentation occurs at night-time temperatures of 8 to 15 °C and 20 °C. Moderate–low outdoor temperatures stimulate the accumulation of
β,β-xanthophylls and C
30 apocarotenoids, which are responsible for the orange colour of citrus fruits and the expression of carotenogenesis-related genes. However, chlorophyll decomposition is delayed in tropical regions under high temperatures (above 25 °C), and there is no characteristic increase in carotenoids [
19]. Therefore, in the present study, the factor governing the delayed colouration under the increased-temperature treatments continued until harvest, which did not promote chlorophyll decomposition. It is thought that the expression of genes related to carotenoids to induce colouration of the pericarp was not proper.
Spiegel-Roy and Goldschmidt [
11] mentioned that tropical orange fruits remain mature and marketable only for a short time, after which they rapidly senesce. Li (cited by Zheng et al. [
22] also reported that excessive temperature and humidity are likely to cause early plant senescence, shorten the growth period, increase vulnerability to pests and diseases, and reduce fruit yield and quality.
Moon et al. [
12] reported that spring heating treatments of Shiranuhi mandarin fruits increased the photosynthesis rate of leaves, which positively affected growth and development. Sucrose and nitrogen have the most important effects on citrus pigmentation during ripening on trees. Nitrogen and sugar are inversely related, because colouration during ripening is induced when the nitrogen content is low and the sugar concentration is high [
19]. Therefore, increasing temperatures from spring to harvest promotes vegetative growth such as fruit development, but it is thought that their effects on sugar content, colouration, and yield negatively affect fruit quality and production.
3.2. Effects of Excessively High Temperatures on Fruit Taste
Soluble sugars are related to carbon, and amino acids are an intermediate product of nitrogen metabolism. Soluble sugars are known to increase resistance to abiotic stresses [
22]. Soluble sugars include mainly glucose, fructose, and sucrose. The amount of sucrose, reaching 15% to 18% of the fresh weight in certain mandarin fruits, exceeds that of fructose and glucose [
11]. In our study, the TSS content and the fructose/glucose ratio were lower under the D8 treatment than under the other treatments. Similarly, at the tuber initiation stage of potatoes under elevated CO
2 and high temperature, the content of hexose (glucose + fructose), which is used as a carbon source, decreased, suggesting that sugar content would decrease if high temperatures increased the rate of metabolism and sugar use [
23].
The exposure of fruits on trees to stress and high temperatures during storage was found to be associated with the accumulation of various amino acids associated with glycolysis and the tricarboxylic acid (TCA) cycle [
24]. We also found that the markedly increased Gly, Ser, Gln, Asn, and Asp contents contributed to metabolism in relation to the photorespiratory nitrogen cycle under high-temperature treatments. The increased high temperature during the day increased the total amino acid content, but the increased high temperature at night or during the day/night was similar to that of the control. Therefore, it is thought that increased daytime temperatures have a greater influence on the amino acid content than do increased night-time temperatures. Proline (Pro) is known to act as a compatible osmolyte, stabilizing the structure of proteins and ROS scavengers, and accumulating in response to various abiotic stresses [
25]. In this study, increasing the high temperature during day or night did not increase the Pro content, but increased high temperature at day/night considerably decreased the Pro content.
Increased daytime/night-time temperature significantly increased the bitterness and sour/umami flavour, while the sweetness and sweetness/bitterness markedly decreased. In the sensory evaluation, the taste of the fruits under the high-temperature treatments was considerably reduced compared to those under the control treatment, with the fruits of the former having a plain or slightly abnormal taste.
Bitterness is well known in grapefruits, but there is almost no bitterness in common mandarin fruits. The bitterness taste of citrus can be classified into two categories: bitterness due to naringin derived from flavonoids, and limonin-based bitterness [
26]. However, naringin was not detected in the flavonoid analysis in this study. However, we were unable to analyse the composition of volatile constituents. Therefore, studies on various volatile compounds related to flavour under excessively high temperatures are needed.
The species, cultivar, and genotype of plants have specific optimal temperature ranges for physiological functions that include biosynthesis of secondary metabolites such as phenolics, alkaloids, flavonoids, and terpenoids, and departure from those ranges can affect biomass and biosynthesis of secondary metabolites [
27]. In our study, increased daytime temperature decreased the contents of narirutin and hesperidin in the peel, whereas rising night-time temperature increased the narirutin, nobiletin, and tangeretin contents. However, the rising daytime/night-time temperatures caused an increase in only the nobiletin contents. The increase in daytime temperatures led to a decrease in narirutin, hesperidin, and rutin contents, even in the pulp. The effects of increased temperature during the night and during the day/night on the pulp were similar to those of the peel. Kim et al. [
28] also showed a change in total flavonoid content in response to low-temperature stress in citrus varieties.
Analysis of the TPC in peel and pulp extracts in response to the different temperature treatments (
Table 6) revealed a higher content in the peel than in the pulp. This seemingly occurred because the peel contains more phenolic compounds than the pulp. Sir Elkhatim et al. [
29] analysed the total contents of extracts of peels, seed-containing pulp, and whole fruits of three species. The results showed a significant difference between species and that, compared with the pulp and seeds, peels contain phenolic compounds. According to Kim et al. [
30], more phenolic compounds are present in the peel than in the pulp, and their contents are higher in non-ripened fruits than in mature fruits.
In conclusion, excessive increases in the temperature during the day, night, or day/night negatively affected fruit size, yield, sugar, acids content, and accumulation of secondary metabolites such as polyphenols and flavonoids, but the accumulation of some amino acids was positively affected. Additionally, the antioxidant activity of ROS scavengers was decreased in response to excessive increases in temperature. Therefore, it is necessary to understand the expression levels of genes related to high temperatures through transcriptome studies, which have recently been performed. In addition, excessive increases in temperature during the day, night, or day/night are expected to negatively affect not only fruit quality but also fruit taste. Consequently, it seems necessary to improve facilities that can minimize damage caused by high temperatures, such as implementing shade screens or developing new varieties with strong high temperature resistance in response to rapidly rising temperatures.