Impaginato 233 Adv. Hort. Sci., 2021 35(3): 233­241 DOI: 10.36253/ahsc­9923 Effect of growth temperature levels on photosynthetic ability and fruit quality of ‘KU­PP2’, a new low­chill peach cultivar P. Sikhandakasmita 1 (*), I. Kataoka 1, T. Ogata 2, R. Mochioka 1, K. Beppu 1 1 Graduate School of Agriculture, Kagawa University, 2393 Ikenobe, Miki, Kagawa 761‐0795, Japan. 2 Faculty of Agriculture and Marine Science, Kochi University, 200 Otsu, Nankoku, Kochi 783‐8502, Japan. Key words: forcing culture, high temperature, protected culture, Prunus persica, stress response. Abstract: Temperature is a crucial factor in growing plants in a forcing system. Our goal was to introduce low­chill peach cultivars into a forcing culture for early­season peach production with high fruit quality. However, the effects of growth temperature on plant growth and fruit quality during fruit development of the ‘KU­PP2’ peach cultivar have not yet been evaluated. ‘KU­PP2’ trees were grown in containers and transferred to phytotrons after fruit set in April 2019. The air temperature was set at 20, 25, and 30°C until harvest. Photosynthetic ability, leaf characteristics, and fruit quality under each treat­ ment were determined. Long exposure to lower growth temperatures did not cause a change in leaf characteristics or a reduction in photosynthetic ability and fruit quality in the ‘KU­PP2’ peach cultivar. In contrast, the 30°C was found to be associated with a decrease in leaf size and thickness, stomatal density, photosynthesis, chlorophyll content, and fruit size. Conversely, the high­tem­ perature condition enhanced coloration of the fruit peel and hastened the har­ vesting period, compared with the lower­temperature treatments. These results indicated that long­term exposure to the moderately high temperature of 30°C negatively affected plant growth and fruit productivity through changed leaf characteristics and a disrupted photosynthesis. 1. Introduction Air temperature is a crucial factor that affects fruit production. Excessive high temperatures disrupt normal plant functions such as car­ bon assimilation, respiration, fertilization, cell differentiation, and fruit maturation (Cui et al., 2006; Efeoglu and Terzioglu, 2009; Lin­Wang, 2011; Hao et al., 2019). Previous report indicated that chlorophyll (Chl) a con­ tent, total Chl content, and the Chl a/b ratio in soybeans, which were grown under high temperatures (38/28°C), decreased 7, 3, and 18%, respectively (Hasanuzzaman et al., 2013). Additionally, Sugiura et al. (*) Corresponding author: panawat.sik@gmail.com Citation: SIKHANDAKASMITA P., KATAOKA I., OGATA T., MOCHIOKA R., BEPPU K., 2021 ­ Effect of growth temperature levels on photosynthetic ability and fruit quality of ‘KU‐PP2’, a new low‐chill peach cultivar. ­ Adv. Hort. Sci., 35(3): 233­241 Copyright: © 2021 Sikhandakasmita P., Kataoka I., Ogata T., Mochioka R., Beppu K. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 19 October 2020 Accepted for publication 10 June 2021 AHS Advances in Horticultural Science https://doi.org/10.36253/ahsc-9923 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2021 35(3): 233­241 234 (2003) showed that higher temperatures significantly change the fruit quality of apple. A reduction in acid concentration and softening of fruit flesh were observed resulting from exposure to high tempera­ tures during fruit development. The effect of elevat­ ed temperatures on plants differs depending on the stages of development and timescale. Continuing heat stress can lead to slowing of growth and devel­ opment and inducing an imbalance in carbohydrate metabolism between photosynthesis and respiration. As a result, carbohydrate reserves decline, leading of yield loss, and possibly plant death (Hall, 1992; Wahid et al., 2007). Photosynthesis comprises a few principal compo­ nents that are highly sensitive to temperature: pho­ tosynthetic pigments, electron transport chain, Photosystem I (PS I), and Photosystem II (PS II). The decline in photosynthesis under high­temperature conditions results from inhibition of the redox reac­ tion and metabolic pathways occurring in PS I, PS II, the cytochrome complex, and photosynthetic enzyme activities (Taiz and Zeiger, 2006). Moreover, elevated temperatures can also affect photosynthesis via physical processes. Previous studies have shown that heat stress is involved with leaf water status, leaf gas exchange, and stomatal conductance (gsw) caused by changes in hydraulic conductance (Fredeen and Sage, 1999; Greer and Weedon, 2012). Under high­temperature conditions, intercellular CO2 concentration in leaves frequently declines because of stomatal closure and reduced CO2 uptake and transport, leading to impaired photosynthetic CO2 assimilation (Centritto et al., 2001). ‘KU­PP2’ is a new yellow flesh peach with a low­ chilling requirement that produce excellent yield and high fruit quality. It was bred and released in 2016 for use in subtropical regions and particularly for use in forcing culture system to expand the harvesting season of fresh peach (Manabe et al. , 2015). Understanding the effect of growth temperatures on ‘KU­PP2’ peach trees is crucial for optimizing plant growth, physiological functioning, and increasing pro­ ductivity. The effect of chilling accumulation and heating temperatures on bud burst and flowering of ‘KU­PP2’ have been clarified. However, the influence of temperature during fruit development on low­chill peach cultivars has not been elucidated. Previous studies on Japanese high­chill peach cultivars indicat­ ed that high temperatures dramatically hasten fruit growth and the onset of fruit maturation (Sugiura et al., 2003; Hayama et al., 2007). In addition, optimal heating could save energy costs for plant production in heated plastic houses. Therefore, the aim of this study was to investigate the effect of growth temper­ ature during fruit development on plant physiology and to determine the optimal growth temperature for plant growth, which can enhance fruit quality of the low­chill peaches under controlled conditions. Additionally, the knowledge gained could be used to design a heating program and cultivation manage­ ment practices for growing the low­chill peach trees in plastic houses. 2. Materials and Methods Plant materials This experiment was conducted at the research field of the Faculty of Agriculture, Kagawa University, which is located in southwest Japan. Six healthy and uniform of seven­year­old ‘KU­PP2’ peach trees were selected for this study. All plants were grafted onto ‘Tsukuba 1 Gou’ peach rootstock and planted in con­ tainers. ‘KU­PP2’ flowers were hand­pollinated with fresh pollen from another ‘KU­PP2’ tree. Three weeks after pollination, two plants were transferred to each temperature regimes and the fruits were thinned by hand to 6­7 fruits per tree. The air temperature in the phytotrons was set at 20, 25, and 30°C during the experimental period from 13 April to 8 July 2019. Cultural practices and fertilization were performed according to standard peach growing practices in Japan (Sugiura et al., 2003). Leaf morphology and anatomy observation Leaf length and width were measured for five mature leaves per tree for each treatment using a digital caliper at the end of the experiment. Leaf width was measured across the widest part of the leaf. Five fully expanded leaves were collected and weighed immediately to determine fresh weight. These leaves were dried in a hot air oven at 80°C and weighed after 72 h of drying to determine their dry weight (Fanourakis et al., 2017). Leaf dry matter (DM) was calculated as the ratio between dry mass and fresh mass. DM was expressed as the percentage of fresh weight For anatomical analysis, five leaf sam­ ples from each plant were collected and preserved in formalin­acetic acid­alcohol (FAA; formaldehyde 1:acetic acid 1: 99.5% ethanol 9:deionized water 9) solution. Cross­sections were made using a rotary microtome at a thickness of 5 µm. The cross­sections Sikhandakasmita et al. ‐ Effect of temperature on the low‐chill peach growth 235 of samples were observed and photographed using a light microscope equipped with a microscope camera (Olympus DP­25, Olympus Co. Ltd., Japan). The fol­ lowing anatomical characteristics were measured: the thickness of the adaxial and abaxial epidermis, spongy mesophyll, and palisade cells, as well as the number of stomata per square millimeter. Evaluations of chlorophyll content and SPAD value The Chl content and SPAD value of five mature leaves from each tree were analyzed during the har­ vesting period. SPAD values were measured using a portable chlorophyll meter (SPAD­502, Minolta, Japan). Chlorophyll in the same leaves was analyzed as described by Lichtenthaler and Wellburn (1983). Leaf disks (2.5 cm2 per disk) were homogenized with 10 mL of cold 95% acetone and incubated at 4°C in darkness for 3 h. These mixtures were centrifuged at 3,500 rpm for 10 min. After centrifugation, absorbance of the supernatants was determined using the spectrophotometer. The optical density for the blank and the mixtures were measured at 645 and 663 nm, respectively. These absorbance values were used to calculate Chl a, Chl b, and total chloro­ phyll (Chl a+b) and expressed as mg L­1. Leaf gas exchange measurement Photosynthetic gas exchange was measured using a Portable Photosynthesis System (LI­6800; LI­COR Biosciences, Lincoln, NE, USA) from 9:00 to 12:00. The rate of net CO2 assimilation, stomatal conduc­ tance, transpiration, and intercellular CO2 concentra­ tion were measured weekly until the end of the experiment. Ten newest fully expanded leaves, which were outside of the canopy and fully exposed to sun­ light, were randomly selected and used for the mea­ surements. The reference CO2 concentration and flow rate inside the chamber were maintained at 400 µmol mol­1 and 800 µmol m­2 s­1, respectively. Photosynthetically active radiation (PAR) was set to 1,200 µmol s­1. The chamber temperature was com­ parable to the growth temperatures, and relative humidity (RH) was kept at 60% (Marchi et al., 2008). The data were recorded at a steady state, in which gas exchange parameters were stable. Fruit quality assessment Five fruits per treatment were collected on the commercial harvest date for phytochemical analysis. After the harvest, all fruits were immediately trans­ ferred to the laboratory and weighed. Flesh firmness and total soluble solids (TSS) were measured from two opposite fruit cheeks. Flesh firmness was deter­ mined using a manual penetrometer with a 4.5­mm tip. TSS was measured using a digital refractometer (PR­101α; Atago Co. Ltd., Japan) and were expressed as degree Brix (°Brix). Titratable acidity (g L­1 of malic acid) was determined by titrating fruit juices with 0.05 mol L­1 of sodium hydroxide (NaOH) using Acidity Titrator (TA­72; DKK­TOA Co. Ltd., Japan). The fruit development period was calculated as the days from full bloom to first commercial harvest. Fruit col­ oring was estimated visually according to a scale from 1 (none) to 9 (hiding ground color) using ECPGR priority descriptors for peach (UPOV, 2010). The fruit coloring was expressed as the percentage of over color extent. Statistical analysis All data from each treatment were subjected to analysis of variance (ANOVA) using the Statistical Analysis System (SAS) university edition (SAS Institute Inc., Cary, NC). The differences between means were separated by Tukey’s honestly significant difference (HSD) test at p<0.05. The results were expressed by means followed by the standard errors. 3. Results Leaf morphology and anatomy response to growth temperature At the end of the experiment, the significant dif­ ferences in leaf dimensions (p < 0.0001) and dry mat­ ter (p<0.0001) between growth temperature levels were observed (Table 1). ‘KU­PP2’ peach trees that were forced at 25°C had the longest leaf length, fol­ lowed by the 20°C and the 30°C treatments, while the leaf width of each treatment was comparable (p= 0.0864). The stomatal density increased by 7% with the increase in growth temperature from 20 to 25°C and reached its maximum value at 25°C. However, raising the growth temperature from 25 to 30°C sig­ nificantly diminished stomatal density by 32%. In contrast, the leaf dry matter content slightly increased when the growth temperature increased. Compared with 20 and 25°C, the 30°C treatment increased leaf dry matter by a mean value of 5% FW. The growth temperatures not only changed the leaf morphological characteristics but also affected leaf anatomical traits (Table 1). The higher tempera­ ture significantly decreased the thickness of leaves, palisade mesophyll, and spongy mesophyll (p < 0.0001). The leaves that were forced at the highest growth temperature (30°C) were thinner than those Adv. Hort. Sci., 2021 35(3): 233­241 236 from the trees grown at 25 and 20°C, as well as pal­ isade and spongy mesophyll layers. On the other hand, the different growing temperatures did not sig­ nificantly change the adaxial and abaxial epidermis thickness (p= 0.7828 and p= 0.4418, respectively) throughout the temperature treatments. Figure 1 shows the light microscopy pictures of leaf cross­sec­ tions for all temperature treatments, measured at the end of treatment. SPAD values, chlorophyll contents, photosynthetic rate, and gas exchange parameters Figure 2 shows the high­temperature conditions caused a reduction in SPAD values (p= 0.0206) and loss of Chl content, especially Chl a (p= 0.0029) and Chl a+b (p= 0.0133). The SPAD reading for the 30°C treatment showed decreases by 12.5%, compared with the 20°C treatment. However, the SPAD values for the 20 and 30°C treatments were not significantly different from that of the 25°C treatment. Similarly, the Chl content decreased when exposed to an increasing temperature compared with the 20°C treatment. The maximum reduction in Chl a content (22.3%) occurred with 30°C, the Chl a+b concentra­ tion for 30°C decreased by 20.1%, while the Chl b content was not significantly affected (p= 0.3494). The responses of the net photosynthetic rate (Pn) and the gas exchange parameters to growth temper­ ature differed significantly depending on the levels and duration of the temperature treatments (Fig. 3). One week after temperature treatment started, the Pn for all treatments increased considerably Fig. 1 ­ Anatomical comparison of leaf cross­section of ‘KU­PP2’ peach trees under (A) 20°C, (B) 25°C and (C) 30°C at the end of the experiment. The cross­sections of samples were observed and photographed under a light micro­ scope. Ep (ad) = adaxial epidermis; Pa = palisade mesophyll layer; Sp = spongy mesophyll layer; and Ep (ab) = abaxial epidermis. These pictures were taken on 15 November 2019. Fig. 2 ­ SPAD value (A) and chlorophyll content (B) in response to growth temperature treatments. Data represent means ± standard error (n= 5). Different letters indicate significant differences according to Tukey’s test (p < 0.05) and NS denotes non­significant. z Data are mean values ± standard errors (n = 10). The different lowercase letters within the same row indicate significant differences at p ≤ 0.05 (Tukey’s test). Table 1 ­ Leaf morphological and anatomical characteristics, percentage of leaf dry matter (DM), and stomatal density of the ‘KU­PP2’ peach cultivar at the end of the experiment. The peach trees were grown under three growing temperatures (20, 25, and 30°C) Parameter Growth temperature p‐value 20°C 25°C 30°C Leaf length (cm) 17.1 ± 0.6 a z 18.6 ± 0.2 a 13.7 ± 0.5 b < 0.0001 Leaf width (cm) 4.6 ± 0.1 4.5 ± 0.1 3.9 ± 0.3 0.0864 Percentage of leaf dry matter (% FW) 44.9 ± 1.35 b 44.5 ± 0.46 b 50.8 ± 0.56 a < 0.0001 Leaf thickness (µm) 58 ± 1.6 a 41 ± 1.0 b 37 ± 1.1 b < 0.0001 Adaxial epidermis thickness (µm) 6 ± 0.3 5 ± 0.3 5 ± 0.5 0.7828 Abaxial epidermis thickness (µm) 4 ± 0.3 4 ± 0.4 3 ± 0.2 0.4418 Palisade thickness (µm) 28 ± 0.7 a 18 ± 0.4 b 15 ± 0.2 c < 0.0001 Spongy thickness (µm) 20 ± 0.5 a 13 ± 0.6 b 14 ± 0.6 b < 0.0001 Stomatal density (no. mm−2) 242 ± 2 b 260 ± 3 a 176 ± 4 c < 0.0001 Sikhandakasmita et al. ‐ Effect of temperature on the low‐chill peach growth 237 (p<0.0001). The Pn of the 30°C treatment was higher than the other treatments in this period (p=0.005). Subsequently, the Pn of the 20 and 25°C treatments steadily increased and remained stable at a higher level than at the beginning of treatment until the harvesting period. Conversely, the Pn of the 30°C treatment dramatically declined in the second week (p=0.0007) and after that gradually decreased and reached its lowest level in the eighth week (p<0.0001) after temperature treatment started (Fig. 3A). The average Pn values of the mature leaves under the 20°C treatment was higher than those in the 25 and 30°C treatments by 12.8 and 47.7%, respectively (p<0.0001). The changes in stomatal conductance (gsw) of each treatment were similar to those of the Pn values. The maximum gsw for 30°C was observed in the first week after the beginning of treatment while the peak gsw for 20 and 25°C occurred in the third week (Fig. 3B). The averages of gsw in both lower­temperature treatments were not different (p= 0.2578). The gsw of the higher­tempera­ ture treatment rapidly decreased in the second (p<0.0001) and eighth weeks (p= 0.02) after treat­ ment started. Similarly, the peak of leaf internal CO2 concentration (Ci) was observed one week after the beginning of treatment (Fig. 3C). A higher growth temperature had greater effects on Ci, with a consid­ erable reduction in Ci occurring twice; in the second and eighth weeks after treatment started (p<0.0001). The lower growth temperatures (20 and 25°C) had comparative effects on the values of transpiration rate (E). The mature leaf E under the 20 and 25°C treatments declined more slowly than under the 30°C treatment, with the average E for the lower temperatures (20 and 25 °C) being higher than that for the high­temperature treatment by 30−35% (p = 0.004; Fig. 3D). Effect of growth temperature on fruit quality indexes The morphological characteristics and chemical compositions of the ripe fruit are shown in Table 2. The results indicated that a high growth temperature strongly affected only the fruit morphological charac­ teristics (p=<0.0001) of the ‘KU­PP2’ fruit and fruit weight (Table 2). However, significant differences in fruit shape (p = 0.0631) and chemical compositions of the fruit (p = 0.0881) were not found. An increase in growth temperature decreased fruit weight, fruit diameter, and fruit length. As shown in figure 4, there were significant contrasts in skin coloration for the ‘KU­PP2’ peaches with the different treatments. During the harvesting period, the fruit from the 30°C treatment showed a higher level of red coloration than the fruit from the 20 and 25°C treatments, indi­ cating that increasing the temperature could acceler­ ate the reddening of the fruit skin. Further, at 30°C, the fruit development period became shorter than under the 20 and 25°C conditions, with maturation occurring 14 days earlier. 4. Discussion and Conclusions Long­term exposure to a moderate high­tempera­ ture regime (30°C) can result in cellular and physio­ Fig. 3 ­ Effect of growth temperatures on (A) leaf net photo­ synthesis, (B) stomatal conductance, (C) leaf internal CO2 concentration, and (D) transpiration rate of the ‘KU­PP2’ peach trees. Data represent means ± standard error (n= 5). 238 Adv. Hort. Sci., 2021 35(3): 233­241 logical adaptation of ‘KU­PP2’. The responses of the peach trees to a high growing temperature could breakdown Chl, change leaf structure, reduce Pn, has­ ten fruit maturity, and could further explain the decrease in fruit quality under high temperatures in the present study. Similar to previous studies, high­ temperature conditions induced closure of the stom­ ata and generation of reactive oxygen species (ROS), damaged chloroplast structure and PS II and decreased photosynthetic pigments and enzyme activities (Takahashi and Murata, 2006; Ashraf and Harris, 2013; Chen et al., 2017; Jumrani et al., 2017). Under high­temperature regimes, plants avoid heat damage and reduce excessive energy absorption on their leaves by decreasing leaf size, covering leaf surfaces with a thick waxy cuticle as well as trichome, changing leaf shape, or increasing the number of stomata. Small leaves can also reduce water loss and have less surface area exposed to solar radiation (Hasanuzzaman et al., 2013). Plants with thinner leaves and high stomatal densities can evacuate heat to the environment quicker than large leaves. A simi­ lar response to high temperatures was found in this study, in which leaf size and thickness of the leaf blades, including the epidermal and mesophyll layers, decreased. Elevating the temperature from 20 to 25°C increased the stomatal density, but the number of stomata sharply decreased when the growth tem­ perature increased from 25 to 30°C. Previous study found similar results: the stomatal density of blue­ berry decreased when the temperature exceeded the optimum growth temperature (Hao et al., 2019). They suggested that increasing stomatal density may be an efficient strategy for evacuating more heat by evaporative cooling, but this strategy is inefficient under higher temperatures (Xu, 2015). It has been reported that a high temperature limits CO2 and H2O diffusion, resulting in increased resistance to gas exchange (Mukohata et al., 1971; Monson et al., 1982). In this study, we found that the transpiration rate (E) of the leaves under the 30°C treatment sharply decreased at four weeks after temperature treatment started, while the E of the leaves under both the 20 and 25°C treatments remained constant or slightly increased. The reduction in E under high­ temperature conditions reflected the low efficiency of leaf cooling. In other words, the convective processes of heat through transpiration were reduced, resulting in excessive leaf temperature above an optimum point. The trees grown at 25°C tended to maintain transpiration cooling by increas­ ing stomatal density, which reduces the negative Table 2 ­ Fruit quality characteristics of the ‘KU­PP2’ peach trees for each growth temperature treatment z Data are mean values ± standard errors (n = 10). The different lowercase letters within the same row indicate significant differences at p≤ 0.05 (Tukey’s test). Parameter Growth temperature p­value 20°C 25°C 30°C Fruit weight (g) 164.34 ± 7.48 a z 131.89 ± 6.16 b 97.83 ± 7.05 c < 0.0001 Fruit cheek diameter (mm) 68.1 ± 1.2 a 62.1 ± 1.4 b 56.2 ± 1.4 c 0.0002 Fruit suture diameter (mm) 69.9 ± 1.3 a 64.0 ± 1.0 b 58.5 ± 1.5 c 0.0002 Fruit length (mm) 59.9 ± 0.7 a 57.7 ± 1.0 a 50.0 ± 0.8 b < 0.0001 Total soluble solids (°Brix) 15.0 ± 0.7 13.8 ± 0.2 13.3 ± 0.6 0.0881 Titratable acidity (g L−1) 0.21 ± 0.07 0.29 ± 0.01 0.24 ± 0.03 0.2331 Over color extent (%) 10–15 b 10–15 b 60–75 a 0.0013 Fruit development period (days) 96 a 91 a 81 b 0.0023 Fig. 4 ­ Effect of growth temperature on the coloration of the ‘KU­PP2’ fruits during the commercial ripening period. Sikhandakasmita et al. ‐ Effect of temperature on the low‐chill peach growth 239 effects of excessive heat on their foliage, leading to the maintaining of high E and Pn. The higher temperature decreased the concentra­ tion of Chl a and Chl a+b. As was also observed in this study, Chl contents have been reported to be sensi­ tive to high­temperature conditions. The decline in Chl pigments may correlate to impaired Chl biosyn­ thesis, exacerbated Chl breakdown, or both. The inhi­ bition of Chl biosynthesis and the increase in Chl degradation under high temperature results from the destruction and construction of several enzymes (Efeoglu and Terzioglu, 2009). Additionally, the reduction in Chl content observed under high tem­ perature is associated with physical damage to thy­ lakoid membranes by excessive ROS accumulation (Halliwell and Gutteridge, 2007). Chl is embedded in the thylakoid membranes; therefore, damage to these membranes could result in Chl loss (Mathur et al., 2014; Chen et al., 2017; Jumrani et al., 2017). The imbalance between Chl biosynthesis and degradation disrupts the photosynthesis apparatus resulting in decreased photosynthetic efficiency, eventually influ­ encing plant growth and fruit quality (Shanshan et al., 2020). Changes in Pn have been directly linked to the level and duration of high­temperature exposure (Hao et al., 2019). In this study, one week after tem­ perature treatment started, the Pn of ‘KU­PP2’ increased rapidly with the initial rise in growth tem­ perature; as the forcing condition continued, Pn under a moderately high­temperature treatment (30°C) dramatically decreased, whereas the Pn under both the 20 and 25°C conditions steadily increased and remained constant until the harvesting period. The response of Pn to growth temperature can depend on two factors ­ non­stomatal and stomatal (Cui et al., 2006; Chen et al., 2014), which can be indicated by the difference in gsw and Ci patterns (Farquhar and Sharkey, 1982). If gsw decreased or stabilized but Ci increased, the decline in Pn can be attributed to non­stomatal factors. If both gsw and Ci decreased simultaneously, Pn could be ascribed to stomatal factors. In this study, the increase in Pn and Ci at the onset of treatment may result from the increase in enzyme activities in the photosynthetic system catalyzed by high temperatures. Therefore, an increase in Pn in this period could be identified as a non­stomatal factor. Furthermore, the decrease in Pn under the pro­ longed higher­temperature treatment (30°C) can be divided into two periods: 2­7 weeks and 8­13 weeks after the onset of forcing. For 2­7 weeks, the decrease in Pn can be ascribed to a non­stomatal limi­ tation, with gsw significantly decreasing and Ci increasing. The non­stomatal factors play a role in the reduction of Pn in the 2­7 weeks period after tem­ perature treatment started through damage to the structures of the chloroplast, impairment of Chl biosynthesis, and increased Chl degradation. This hypothesis is supported by the reduction in Chl a and Chl a+b observed in this study. With exposure to forcing conditions over an extended period, Pn, Ci, and gsw of the 30°C treatment gradually decreased and reached their lowest levels in the eighth week after temperature treatment started, indicating that Pn in this period might be limited by stomatal factors through changes in stomatal density and modified leaf morphological and anatomical characteristics. Our study showed that the size and thickness of the leaves, including the epidermal and mesophyll layers, decreased with the elevated growth temperature, and thus led to the decline in Pn as stomatal limita­ tions. The differences in fruit morphological characteris­ tics, such as fruit weight and fruit size of the trees under high­temperature conditions, might be associ­ ated with the decline in the fruit development period and Pn. Previous studies reported that the relation­ ships between fruit development period (FDP) and fruit weight and diameter were observed in apple and peach (Sugiura et al., 2013; Giovannelli et al., 2014). In the 30°C treatment, fruit size was lower than those in the 20 and 25°C treatments, which was expected according to the length of their FDP. Additionally, previous studies indicated that a low Pn causes a steep reduction in fruit size because most of the energy used in fruit development is generated via photosynthesis during the year (Pavel and DeJong, 1993; Grossman and DeJong, 1995) Similarly, Lopez and DeJong (2007) reported that high temperature during fruit development increases the potential of fruit growth without enough resources to subsidize fruit growth, resulting in smaller fruit size. High tem­ peratures not only depress photosynthesis but also increase leaf respiration. Plants grown under high­ temperature conditions may consume much more energy because of increased leaf respiration caused by increased temperatures (Corelli­Grappadelli and Lakso, 2004; Hao et al., 2019). This result is support­ ed by the increase in both the number and size of mitochondria in Arabidopsis thaliana, indicating that more starch and soluble sugar are consumed by leaf respiration and rapid growth because of increased temperature (Jin et al., 2011). Hence, the reduction in fruit size of ‘KU­PP2’ grown under high tempera­ tures may be supported by the above conclusion. In this study, we found that the red coloration in ‘KU­PP2’ peel at 30°C was higher than those at 20 and 25 °C. Previous study showed the red coloration in plum (P. salicina Lindl.) peel increases under high­ temperature conditions (35°C) (Junping et al., 2017). Conversely, the biosynthesis of anthocyanin in grape and apple is suppressed by high temperatures (Lin­ Wang, 2011; Mori et al., 2017). Junping et al. (2017) showed that high temperatures can stimulate red skin coloration in plum by increasing respiration and ethylene production. Long­term forcing under high­ temperature conditions may increase the respiration rate in ‘KU­PP2’ peach fruits, and hence enhance red coloration in the fruit peel. Moreover, the develop­ ment of red coloration in peach fruit skin is positively related to light conditions (Corelli­Grappadelli and Coston, 1991; Kataoka and Beppu, 2004). Previous study showed that ‘Redhaven’ peach fruits that develop in the shade have less red coloration than those that develop in full sunlight (Erez and Flore, 1986). In our study, the fruit grown under the 30°C treatment had smaller leaves, leading to a decrease in canopy shade. Thus, the peach fruits grown under the 30°C treatment were exposed to more sunlight, which might result in higher red skin coloration. In conclusion, this study illustrated the effect of growth temperature on plant development and fruit quality of ‘KU­PP2’ peach trees. Air temperatures directly affect leaf morphology, leaf anatomy, and the photosynthetic ability of plants. The decline in carbon assimilation due to exposure to excessive temperatures could diminish the plant’s ability to efficiently support fruit development, resulting in low yield and poor fruit quality. All these data show high­ temperature stress in the ‘KU­PP2’ peach cultivar caused by long­term exposure to moderately high temperatures. Therefore, a better understanding of plant adaptability to high temperatures is crucial for growing low­chill peach cultivars in plastic houses with a heating system. Acknowledgements This research was funded by Japan Society for the Promotion of Science (JSPS) KAKENHI, grant number 18K05621. Adv. Hort. Sci., 2021 35(3): 233­241 240 References ASHRAF A., HARRIS P.J.C., 2013 ­ Photosynthesis under stressful environments: an overview. ­ Photosynthetica., 51: 163­190. CENTRITTO M., BRILLI F., FODALE R., LORETO F., 2001 ­ Different sensitivity of isoprene emission, respiration and photosynthesis to high growth temperature cou‐ pled with drought stress in black poplar (Populus nigra) saplings. ­ Tree Physiol., 31: 275­286. CHEN T.W., HENKE M., DE VISSER P.H.B. BUCK­SORLIN G., WIECHERS D., KAHLEN K., STUTZEL H., 2014 ­ What is the most prominent factor limiting photosynthesis in different layers of a greenhouse cucumber canopy? ­ Ann. Bot., 114: 677­688. CHEN Y.E., SU Y.Q., ZHANG C.M., MA J., MAO H.T., YANG Z.H., YUAN M., ZHANG Z.W., YUAN S., ZHANG H.Y., 2017 ­ Comparison of photosynthetic characteristics and antioxidant systems in different wheat strains. ­ J. Plant Growth Regul., 37(2): 347­359. CORELLI­GRAPPADELLI L., COSTON D.C., 1991 ­ Thinning pattern and light environment in peach tree canopies influences fruit quality. ­ HortScience., 26: 1464­1466. CORELLI­GRAPPADELLI L., LAKSO A.N., 2004 ­ Fruit devel‐ opment in deciduous tree crops as affected by physio‐ logical factors and environmental conditions. ­ Acta Horticulturae, 636: 425­441. CUI L.J., LI J.L., FAN Y.M., XU S., ZHANG Z., 2006 ­ High tem‐ perature effects on photosynthesis, PSII functionality and antioxidant activity of two Festuca arundinacea cultivars with different heat susceptibility. ­ Bot. Stud., 47: 61­69. EFEOGLU B., TERZIOGLU S., 2009 ­ Photosynthetic respons‐ es of two wheat varieties to high temperature. ­ Eur. Asia J. BioSci., 3: 97­106. EREZ A., FLORE J.A., 1986 ­ The quantitative effect of solar radiation on ‘Redhaven’ peach fruit skin color. ­ HortScience., 21: 1424­1426. FANOURAKIS D., HYLDGAARD B., GIDAY H., BOURANIS D., KÖRNER O., NIELSEN K.L., OTTOSEN C.O., 2017 ­ Differential effects of elevated air humidity on stomatal closing ability of Kalanchoë blossfeldiana between the C3 and CAM states. ­ Environ. Exp. Bot., 143: 115­124. FARQUHAR G.D., SHARKEY T.D., 1982 ­ Stomatal conduc‐ tance and photosynthesis. ­ Annu. Rev. Plant Physiol., 33: 317­345. FREDEEN A.L., SAGE R.F., 1999 ­ Temperature and humidity effects on branchlet gas exchange in white spruce: an explanation for the increase in transpiration with branchlet temperature. ­ Trees Struct. Funct., 14: 161­ 168. GIOVANNELLI C., BOUZO C., RIBERO G., CASTRO D., MICHELOUD N., GARIGLIO N., 2014 ­ External fruit quality and harvest time of low‐chill peach and nec‐ tarine varieties at Santa Fe, Argentina. ­ Aust. J. Basic Appl. Sci., 8(1): 427­433. Sikhandakasmita et al. ‐ Effect of temperature on the low‐chill peach growth 241 GREER D.H., WEEDON M.M., 2012 ­ Modelling photosyn‐ thetic responses to temperature of grapevine (Vitis vinifera cv. Semillon) leaves on vines grown in a hot cli‐ mate. ­ Plant Cell Environ., 35: 1050­1064. GROSSMAN Y.L., DEJONG T.M., 1995 ­ Maximum fruit growth potential and seasonal patterns of resource dynamics during peach growth. ­ Ann. Bot., 75: 553­560. HALL A.E., 1992 ­ Breeding for heat tolerance. ­ Plant Breed. Rev., 10: 129­168. HALLIWELL B., GUTTERIDGE J., 2007 ­ Free radicals in bio‐ logy and medicine. ­ Oxford University Press, Oxford, UK, pp. 944. HAO L., GUO L., LI R., CHENG Y., HUANG L., ZHOU H., XU M., LI F., ZHANG X., ZHENG YH., 2019 ­ Responses of photosynthesis to high temperature stress associated with changes in leaf structure and biochemistry of blue‐ berry (Vaccinium corymbosum L.). ­ Sci. Hortic., 246: 251­264. HASANUZZAMAN M., NAHAR K., ALAM M.Md., ROYCHOWDHURY R., FUJITA M., 2013 ­ Physiological, biochemical, and molecular mechanisms of heat toler‐ ance in plants. ­ Int. J. Mol. Sci.,14(5): 9643­9684. HAYAMA H., FUJIMARU O., IWATANI A., ITO A., SAKAMOTO D., OKADA S., KASHIMURA Y., 2007 ­ Influences of temperature during fruit growing season on fruit development of ‘Akatsuki’ peach. ­ Hort. Res. (Japan), 6(2): 201­207. JIN B., WANG L., WANG J., JIANG K., WANG Y., JIANG X., NI C., WANG Y., TENG N., 2011 ­ The effect of experimen‐ tal warming on leaf functional traits, leaf structure and leaf biochemistry in Arabidopsis thaliana. ­ BMC Plant. Biol., 11: 35. JUMRANI K., BHATIA V.S., PANDEY G.P., 2017 ­ Impact of elevated temperatures on specific leaf weight, stomatal density, photosynthesis, and chlorophyll fluorescence in soybean. ­ Photosynth. Res., 131: 333­350. JUNPING N., GUAJING Z., WENTING Z., VASILIJ G., SHAN S., JINZHENG W., PENGMIN L., FENGWANG M., 2017 ­ Anthocyanin concentration depends on the counterbal‐ ance between its synthesis and degradation in plum fruit at high temperature. ­ Sci. Rep., 7: 7684. KATAOKA I., BEPPU K., 2004 ­ UV irradiance increases development of red skin color and anthocyanins in ‘Hakuho’ peach. ­ HortScience., 39(6): 1234­1237. LICHTENTHALER H., WELLBURN A., 1983 ­ Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. ­ Biochem. Soc. Trans., 11: 591­592. LIN­WANG K., 2011 ­ High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex. ­ Plant Cell. Environ., 34: 1176­1190. LOPEZ G., DEJONG T.M., 2007 ­ Spring temperatures have a major effect on early stages of peach fruit growth. ­ J. Hortic. Sci. Biotech., 82(4): 507­512. MANABE T., BEPPU K., KATAOKA I., 2015 ­ New lower‐chill‐ ing peach cultivar with yellow flesh, ‘KU‐PP2’. ­ Hortic. Res. (Japan), 14: 287. MARCHI S., TOGNETTI R., MINNOCCI A., BORGHI M., SEBASTIANI L., 2008 ­ Variation in mesophyll anatomy and photosynthetic capacity during leaf development in a deciduous mesophyte fruit tree (Prunus persica) and an evergreen sclerophyllous Mediterranean shrub (Olea europaea). ­ Trees., 22: 559­571. MATHUR S., AGRAWAL D., JAJOO A., 2014 ­ Photosynthesis: response to high temperature stress. ­ J. Photochem. Photobiol. B. Biol., 137: 116­126. MONSON R.K., STIDHAM M.A., WILLIAMS G.J., EDWARDS G.E., 1982 ­ Temperature dependence of photosynthe‐ sis in Agropyron smithii Rydb. I. factors affecting net CO2, uptake in intact leaves and contribution from ribu‐ lose‐1,5‐bisphosphate carboxylase measured in vivo and in vitro. ­ Plant Physiol., 69: 921­928. MORI K., GOTO­YAMAMOTO N., KITAYAMA M., HASHIZUME K., 2017 ­ Loss of anthocyanins in red‐wine grape under high temperature. ­ J. Exp. Bot., 58: 1935­ 1945. MUKOHATA Y., MITSUDO M., KAKUMOTO S., HIGASHIDA M., 1971 ­ Biophysical studies in subcellular particles. V. Effects of temperature on the ferricyanide‐Hill reaction, the light‐induced pH shift and the light scattering response of isolated spinach chloroplasts. ­ Plant Cell Physiol., 12: 866­880. PAVEL E.W., DEJONG T.M., 1993 ­ Relative growth rate and its relationship to compositional changes of nonstruc‐ tural carbohydrates in the mesocarp of developing peach fruits. ­ J. Amer. Soc. Hort. Sci., 118: 503­508. SHANSHAN H., YANFEI D., CHENG Z., 2020 ­ Sensitivity and responses of chloroplasts to heat stress in plants. ­ Front. Plant Sci., 11: 375. SUGIURA T., OGAWA H., FUKUDA N., MORIGUCHI T., 2013 ­ Change in the taste and textural attributes of apples in response to climate change. ­ Sci. Rep., 3: 2418. SUGIURA T., TAKADA N., KURODA H., SUGIURA H., 2003 ­ Influence of temperature in young fruit stage on growth, development, and cell division of ‘Hakuho’ peach fruits. ­ J. Jpn. Soc. Hort. Sci., 72(2): 340. TAIZ L., ZEIGER E., 2006 ­ Plant physiology. ­ 4th ed., Sinauer Associates, Inc., Sunderland, MA, USA. TAKAHASHI S., MURATA N., 2006 ­ Glycerate‐3‐phosphate, produced by CO2 fixation in the Calvin cycle, is critical for synthesis of the D1 protein of photosystem II. ­ Biochim. Biophys. Acta, 1757: 198­205. UPOV, 2010 ­ Guidelines for the conduct of tests for dis‐ tinctness, uniformity and stability. Peach. TG/53/7. ­ International Union for the Protection of New Varieties of Plants, Geneva, Switzerland, pp. 18. WAHID A., GELANI S., ASHARF M., FOOLAD M.R., 2007 ­ Heat tolerance in plants: an overview. ­ Environ. Exp. Bot., 61: 199­223. XU M., 2015 ­ The optimal atmospheric CO2 concentration for the growth of winter wheat (Triticum aestivum). ­ J. Plant. Physiol., 184: 89­97.