The Effect of Short-Term Temperature Pretreatments on Sugars, Organic Acids, and Amino Acids Metabolism in Valencia Orange Fruit

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Introduction
Citrus is one of the most popular and important fruits in the world because of its high nutritional and commercial value. Citrus fruits are rich in organic acids (particularly citric acid and malic acid), solute sugars (such as sucrose and fructose), and multiple amino acids that give them excellent favor [1]. With the development of the citrus industry, the improvement and maintenance of fruit quality have been attracting more and more attention. Postharvest treatments are considered important strategies to maintain the fruit quality and extend the shelf life of citrus fruit [2].
To date, various preservation methods, including physical preservation and chemical preservation, have been widely used in the industry [3][4][5]. Physical preservation methods, such as temperature treatments, show excellent efects on the maintenance of nutrition and natural favors and are more environmentally friendly and safe approaches compared with chemical preservation methods [6].
Temperature treatments have been widely used as a preharvest and postharvest strategy in fruit preservation. Lowtemperature (LT) treatment can efectively repress quality deterioration and delay fruit senescence, thereby extending the shelf life of fresh fruit [7][8][9]. Short-term LT, or precooling treatment, is a typical method to remove the respiratory heat and regulate fruit metabolism and has a good efect on maintaining fruit quality during postharvest [10,11]. Recently, transcriptomic and metabolomic analyses revealed that appropriate LT or cold storage improved the inner and external quality via modulating multiple metabolic pathways [12,13].
On the other hand, short-term high temperature (HT) treatment, such as hot water, hot steam, and hot air treatment, is another important physical preservation method and has become a necessary and efective pretreatment method before the long-term storage of fresh fruit. It has been reported to play an important role in maintaining postharvest quality and extending the shelf life of fresh fruit [14,15]. HT treatment can not only repress postharvest respiration and water loss but also maintain the content of favor metabolites such as sugar and organic acid [16][17][18][19][20][21].
Delaying the quality deterioration of citrus fruit is the primary goal throughout the storage. As is known, the lateripening citrus fruit will reach commercial maturity in the spring or summer seasons of rising temperatures. Te metabolic activity of citrus fruit is very high, which causes difculty in maintaining the postharvest quality of fruit [22]. Meanwhile, preharvest treatments (such as heat treatment) have been determined to efectively maintain or improve fruit quality during long storage [20,23]. However, the information about the quality changes and direct efects of short-term temperature treatments on late-ripening citrus fruit is limited. Terefore, this study aimed to evaluate the efects of diferent temperature treatments on the fruit quality of late-ripening citrus and provide more information on the improvement of physical preservation.

Plant Materials.
Valencia orange (citrus sinensis) fruits were harvested at the commercially mature stage (April) from the orchard of Zigui Country in Yichang City, Hubei Province, China. After the harvest, the fruits with obvious damage were removed.

Experimental Design and Treatments.
Valencia orange fruits of uniform size and homogeneous color were selected for experiments. For cold or heat shock treatments, Valencia orange fruits were stored in storage chambers (LRH-70F, Yiheng, China) with diferent parameters (relative humidity, 85%-90%; temperature, 6°C (low temperature, LT), 20°C (room temperature, CK), and 40°C (high temperature, HT)). Te storage chambers with low and high temperatures were used to simulate the cold and heat shock treatments, respectively. Te samples treated with diferent temperatures were conducted at 6 and 24 hours after treatment (HAT), respectively. 80 fruits of uniform size and color and free of visible injury or blemishes were used in each treatment. Tree replications, each containing 10 fruits, were analyzed and measurements were performed. Te favedo (outer colored part of the peel), albedo (inside colorless part of the peel), and pulp were, respectively, sampled, frozen, and homogenized in liquid nitrogen, and kept at −80°C for later analysis.

Extraction of the Primary Metabolite.
Te primary metabolites of diferent tissues in citrus fruit were detected with the approach of GC-MS analysis according to the method as described previously with minor modifcations [24]. Fruit tissues were grounded in liquid nitrogen, and then 0.3 g of diferent samples were used for the following metabolite extraction. Te primary metabolites were extracted with 2700 μL precooled methanol and 300 μL ribitol (2 mg·mL −1 , as an internal standard).

GC-MS Analysis of Primary Metabolite.
After derivatization, each sample was analyzed by GC-MS with the programs described previously [23]. Briefy, each sample was injected into the gas chromatograph onto a fused-silica capillary column (30 m × 0.25 mm i.d., 0.1 μm, Agilent Technologies) with a split ratio of 20 : 1. Te injector temperature was 230°C, and the carrier gas was at a fow rate of 1.2 mL/min. Te column temperature was held at 100°C for 1 min, increased to 184°C with a temperature gradient of 3°C/min, increased to 190°C at 0.5°C/min for 1 min, and increased to 280°C with a temperature gradient of 15°C/min. Te column temperature was held at 280°C for 5 min. Te fow rate of carrier helium (99.999%) gas was 1 mL/min. Total ion current spectra were recorded over a mass range of m/z 45-600 in a scan mode. Te fnal concentration of the metabolite was qualifed using the internal standard (mg/g).
2.6. Statistical Analysis. All data are shown as the (mean ±SD) of one representative experiment. Signifcant diferences between treatments were determined using an ANOVA followed by a Tukey's test. Te partial least squares discriminant analysis (PLS-DA) was performed using the mixOmics package. Te heatmaps and hierarchical clustering were performed using the pheatmap package. Te correlation analysis was performed using R studio software. Figures were drawn using a GraphPad Prism (GraphPad Software, CA, USA).

Efect of Short-Term Temperature Treatments on the Total Sugar and Organic Acid in Fruit
Pulp. Te late-ripening orange fruits, namely Valencia orange fruits, were treated at diferent temperatures. After 24 hours of treatment, no signifcant diferences were observed in fruit appearance between diferent treatments (Figure 1(a)). As shown in Figure 1(b), after 6 hours of treatment, the content of total sugar in the pulp of all treated fruits was similar (35 mg/g to 42 mg/g). After 24 hours, the sugar content in the pulp treated at relatively high temperatures (CK, 44 mg/g; high, 53 mg/g) was higher than that of low (34 mg/g). Meanwhile, the content of total organic acid in the pulp treated at relatively high temperatures (CK and high) was much higher than that treated at low temperatures after 6 and 24 hours. Te content of total organic acid was observed to be lower after 24 hours of treatment with high temperature (HT) compared with that after 6 hours. Besides, the ratio of sugar to acid in fruit treated with HT was much higher at 24 hours but lower at 6 hours compared with other treatments (Figure 1(b)).

Efect of Short-Term Temperature Treatments on Sugar
Content in Fruit Tissues. Te three main forms of soluble sugar in fruits, namely fructose, glucose, and sucrose, were further analyzed. As shown in Figure 2, the content of these sugars in the pulp shared similar trends with the total sugar, and the content of those treated with HT (high temperature) after 24 hours was signifcantly higher than other treatments. In the favedo, fructose and glucose showed the highest content in CK after 6 hours of treatment and the lowest content in CK after 24 hours of treatment. Besides, the content of fructose and glucose in LT treatment was observed to increase over time, while that of them in CK decreased. In albedo, the content of fructose and glucose was lower after 24 hours of LT treatment than that after CK and HT treatments (Figures 2(a) and 2(b)). Notably, the content of sucrose in diferent fruit tissues shared similar trends, and it was highest after 24 hours of HT treatment (Figure 2(c)). Tese results indicated that the efect of diferent temperatures on the three main sugars in the pulp was similar, as well as the sucrose in the peel (favedo and albedo).

Efect of Short-Term Temperature Treatments on the
Organic Acid Content in Fruit Tissues. As the most important organic acid in fruit pulp, citric acid was observed to signifcantly increase under the relatively high temperature (CK and high) and showed an unchanged content under LT treatment (Figure 3(a)). Meanwhile, the malic acid showed similar trends with a total organic acid content, but its content in CK after 6 hours of treatment was signifcantly lower than in the other treatments (Figure 3(b)). Te content of quininic acid was observed to be elevated by HT in the pulp of the fruit (Figure 3(c)). In favedo, citric acid had a lower accumulation (0.01 mg/g to 0.02 mg/g), compared with that in the pulp (more than 2 mg/g to 3 mg/g) (Figure 3(a)). As the main organic acid in favedo, malic acid showed decreased content over time under diferent temperature treatments and had the highest content under LT treatment after 6 hours (Figure 3(b)). Te content of quininic acid was much higher in favedo (0.1 mg/g to 0.2 mg/g) than that in other tissues (less than 0.03 mg/g). In albedo, all these organic acids showed low accumulation (less than 0.1 mg/g). Citric acid and malic acid were observed to be decreased under LT treatment after 24 hours. Besides, the content of quininic acid decreased signifcantly over time in the albedo of fruit (Figure 3(c)). In summary, the changes in the content of citric acid almost accounted for the changes in total organic acid in the pulp. Besides, HT treatment induced the decline of major organic acids in diferent tissues.

Efect of Short-Term Temperature Treatments on Amino
Acid Content in Fruit Tissues. In total, 11 amino acids were detected in this assay, as well as 3 other metabolites. As shown in Figure 4(a), most of these metabolites (particularly glycine, GABA, and Myo-inositol) were signifcantly reduced in the pulp under LT treatment after 24 hours. In the pulp under CK, the contents of aspartic acid, oxoproline, palmitic acid, and stearic acid were observed to accumulate after 24 hours, while GABA and asparagine were decreased.
After HT treatment, valine, alanine, glycine, and Myoinositol showed higher accumulation than those under other treatments. Meanwhile, the content of GABA, proline, and threonine was increased after 24 hours of treatment (Figure 4(a)). In favedo, oxoproline, palmitic acid, and stearic acid were signifcantly accumulated 24 hours under relative higher temperature treatments (CK and high) after whereas, aspartic acid, asparagine, and glutamic acid had high content after 6 hours of treatment followed by obviously decreasing after 24 hours' treatment under LT and CK treatments (Figure 4(b)). Additionally, valine, GABA, and proline showed higher content after 24 hours of HT treatment (Figure 4(b)). In albedo, the of content oxoproline, Myo-inositol, palmitic acid, and stearic acid highly accumulated after 6 hours of HT treatment; however, aspartic acid, asparagine, and glutamic acid shared similar content trends with that in favedo (Figure 4(c)). Te content of several amino acids (such as serine, glycine, and threonine) was much higher than that in other treatments. Notably, GABA was found to signifcantly accumulate after 24 hours   of HT treatment, while decreasing after 24 hours of LT treatment (Figure 4(c)). Together, the LT treatment caused the decline of most amino acids, while the HT treatment contributed to the accumulation of them (particularly GABA and proline) in diferent tissues.

PLS-DA and Correlation Analysis.
To better understand the efect of short-term temperature treatments on fruit metabolites, partial least-squares discrimination analysis (PLS-DA) was used to investigate the diferences in metabolites among diferent treatments. As shown in Figure 5(a), PC1 and PC2 accounted for 24% and 12% of the total variance, respectively. Most samples under the same treatment were clustered together, while the favedo after 6 hours of HT treatment (High_6_FL) and the favedo after 24 hours of LT treatment (Low_24_FL) were found to be clustered with tissues under CK treatment. Besides, there was an obvious time-specifcity under the same temperature treatment. Furthermore, Pearson correlation analysis was constructed to analyze the relationships between diferent metabolites. As shown in Figure 5(b), fructose was positively related to glucose (R 2 � 0.9), and sucrose was closely related to several amino acids. Besides, GABA was observed to be positively related to citric acid and malic acid, which were negatively related to Myo-inositol ( Figure 5(b)). Tese results indicate that temperature treatments have a signifcant efect on the metabolic fow of citrus fruit.

Discussion
Te preharvest or postharvest treatments have become crucial parts of the citrus industry and contribute to the annual supply of citrus fruit. To date, various strategies have been developed and demonstrated to efectively maintain the fruit quality and extend the shelf life [25][26][27][28]. It is worth noting that temperature acts as an essential environmental factor and modulates the respiration and metabolic activities in various metabolism pathways [2,29,30].
As it is known, the temperature has been indicated to be a fundamental factor infuencing the quality of citrus fruit. Maintaining storage temperature or removing feld heat from fresh fruit will decrease the deterioration and senescence processes, which obviously preserves fruit quality [29,31]. Previously, the sweating treatment, which was used to remove the feld heat of fruit, was also found to contribute to maintaining fruit quality and increasing disease resistance [23]. In the present study, short-term LT treatment was also found to maintain the content of main quality components, including solute sugars and organic acids, in the pulp of Valencia orange fruit (Figures 2 and 3). Te slight changes in the content of sugar and organic acid may be mainly due to the low activity of primary metabolism caused by low temperatures [32,33]. Whereas, multiple amino acids (particularly GABA and proline) were observed to show decreased content after short-term LT treatment compared with that after HT treatment (Figure 4). Te degradation of amino acids modulated by LT may account for the changes in amino acid metabolic enzymes and transporters [34,35].
Similar to short-term LT treatment, HT treatment is usually applied as a pretreatment method to treat fresh fruits (such as citrus and apples) before long-term storage [14,36,37]. Recent studies have revealed that HT treatment can modulate metabolic fow by regulating lots of genes and enzyme activity [15,18,38]. Herein, HT treatment was found to elevate the accumulation of three main sugars and several amino acids (such as GABA) in fruit pulp ( Figure 2). Whereas, the content of citric acid and malic acid signifcantly decreased after 24 hours of HT treatment (Figure 3). Similarly, the content of soluble solids in mandarin fruit and persimmon fruit treated with HT also increased, but citric acid showed decreased content [39,40]. As reported previously, the HT treatment stimulated sugar metabolism and induced the accumulation of sugars; meanwhile, it activated the transcription of many genes related to citric degradation [23,38]. Besides, our results showed that HT treatment resulted in a higher sugar-to-acid ratio after 24 hours of treatment and a lower ratio after 6 hours (Figure 1(b)). As reported previously, the diferent efects of HT treatment on fruit quality may account for the treatment duration, the temperature, handling, or even the fruit species [14,41,42]. Hence, to investigate the treatment duration or number of handlings on citrus fruits will maximize the efect of HT treatment on quality improvement and maintenance.
Our study found that several amino acids accumulated in the peel (favedo and albedo) after HT treatment but decreased with LT or CK treatment. Notably, the nonprotein amino, namely GABA, showed the highest content after 24 hours of HT treatment but the lowest content after LT treatment ( Figure 4). In mandarin and peach fruit, GABA was also found to be upregulated after sweating or hot water treatment [23,43]. However, GABA in longan fruit showed increased accumulation after precooling treatments, which was diferent from our results [44]. Studies have shown that GABA is mainly metabolized via the GABA shunt, which is one of the pathways involved in citric acid degradation [45,46]. Hence, the up or downregulated GABA may be due to the specialized primary metabolic activity response to temperature treatments. Notably, GABA has been investigated to be an important signal molecule involved in the regulation of fruit preservation, disease resistance, and plant development [47,48]. Exogenous GABA treatments were also found to improve fruit quality and delay postharvest senescence [24,49,50]. Terefore, it is worth investigating the regulation mechanism of GABA in response to temperature and its potential functions in fruit quality control.

Conclusions
Temperature treatments have a signifcant efect on the citrus fruit quality. In this study, short-term LT treatment contributes to the maintenance of sugars (including fructose, glucose, and sucrose) and citric acid, but decreases the content of several amino acids in the pulp of citrus fruit. HT treatment signifcantly promotes the accumulation of sugars and lots of amino acids but decreases the content of organic acids. Meanwhile, the signaling molecular GABA was upregulated after HT treatment but downregulated after LT Journal of Food Quality treatment. Our results indicated that the short-term temperature treatments afected the metabolic activity in different tissues of citrus fruit, and GABA may play a part in the regulation of fruit quality. Te content of metabolites, metabolic activity, and activation of signal pathways are important factors afecting the fruit's storage quality before long-term storage. Our results provide new insights for the study of temperature-regulated fruit quality.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
Te authors declare that there are no conficts of interest.

Authors' Contributions
Mingfei Zhang oversaw conceptualization, validation, formal analysis, investigation, resources, writing-original draft, and visualization. Resources, investigation, and project administration were done by Rangwei Xu, Guochao Sun, and Yunjiang Cheng. Zhihui Wang was responsible for supervision, project administration, and funding acquisition.