Melatonin Improves Storage Quality of Sweetpotato (Ipomoea batatas) by Inhibiting Sprouting, Weight Loss, and Lignification and Elevating Sweetness

TL;DR

Melatonin improves sweetpotato storage quality by reducing sprouting, weight loss, and lignification while increasing sweetness.

Contribution

This study demonstrates the novel application of melatonin to enhance storage quality of sweetpotato roots.

Findings

Melatonin inhibits sprouting by altering germination-related hormone levels.

Melatonin reduces weight loss and lignification by suppressing respiration.

Melatonin increases sweetness by boosting soluble sugar content through enzyme regulation.

Abstract

It has been well established that exogenous melatonin (MT) improves storage quality of many agricultural products. However, contrasting results have been reported in the regulation of MT with respect to several postharvest parameters, e.g., germination/sprouting and lignification, indicating that roles of MT may vary with plant species or storage environment. Previous studies mainly focus on above-ground organs including fruits, leaves, seedlings and flowers without addressing underground organs such as the storage root (SR) of sweetpotato (Ipomoea batatas). This study showed that spraying 0.5 mM MT solution improved postharvest quality of sweetpotato SRs during 40 d of storage at 15 °C. First, MT treatment inhibited SR sprouting by differentially regulating the content of germination-related hormones, i.e., increasing the content of ABA and JA but decreasing GA content. Second, MT reduced weight loss and lignification by inhibiting respiration as reflected by decreased respiration rate and hexose kinase activity. Third, MT treatment increased soluble sugar content by elevating the activity and expression of sucrose synthase (Sus) since the activities and expressions of invertases (CWIN, CIN and VIN) were inhibited by MT. Simultaneously, inhibited respiration by MT also contributed to increased content of soluble sugar by reducing their expenditure via glycolysis. Additionally, MT increased starch content by inhibiting β-amylase activity and possibly also by increasing Sus activity, which provides a substrate for starch biosynthesis. Finally, MT upregulated the activities of SOD, POD and CAT, which may improve storage quality of SRs by inhibiting senescence and lignification. This study provides an alternative option to maintain the storage quality of sweetpotato.

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1. Introduction

Sweetpotato (Ipomoea batatas) is a dicotyledon crop belonging to the botanical family Convolvulaceae. Due to its strong environmental adaptability and high yield [1], it has become the seventh most important crop in the world after maize, rice, wheat, soybean, potato, cassava and barley [2]. Sweetpotato is mainly cultivated in developing countries, especially those in Asia and Africa [3]. As the largest country in sweetpotato cultivation worldwide, the cultivating area and total production of China amount to 2.37 million hectares and 52 million tons, accounting for 32% and 57% of the whole world’s production, respectively [4]. The demand for sweetpotato in developed countries has also been steadily growing in recent years [5]. The storage root (SR) of sweetpotato is rich in carbohydrates, dietary fiber, vitamins, anthocyanins, carotenoids, flavonoids, and phenolic compounds, thus showing outstanding health-promoting features such as anti-inflammatory, anti-diabetic, anti-hypotensive properties and specific anti-cancer bioactivities [6]. Although sweetpotato is usually consumed as a vegetable in developed countries, it is cultivated as a staple crop in many developing countries and plays an important role in the food security of the world [5].

The storage of sweetpotato not only allows their year-round supply for the consumer but also increases the sugar content (sweetness) of SRs and thus their palatability and marketability [7,8]. However, storage inevitably induces various adverse physicochemical changes, including sprouting, postharvest diseases, weight loss and quality decline [9]. These physicochemical changes become more significant during long-term storage, which results in severe postharvest losses, which account for 20 to 30% of total production [9].

There are several options to alleviate or slow down these changes in sweetpotato. The most widely used method is cold storage at a temperature of 13–16 °C and a relative humidity of 80–95% due to its simplicity and effectiveness [10]. However, the cold storage usually leads to a dramatic increase in storage cost resulting from the establishment of cold storage facilities and the high energy consumption, which is unaffordable for producers in developing countries [9]. Alternatively, several chemicals such as chlorpropham (CIPC), ethylene (ETH) and 1-methylcyclopropene (1-MCP) are employed to extend the storage period of sweetpotato. However, the use of CIPC usually causes environmental pollution and health damage due to its residues in sweetpotato SRs [11]. Although green preservatives ETH and 1-MCP are secure and non-toxic, their roles are cultivar-dependent since different sweetpotato cultivars have different sensitivity to the chemicals [9]. Low dosage of them cannot maintain storage quality effectively, whereas high dosage of them may incur side effects including root disease, increased respiration rate, excessive weight loss and decreased sugar content in sweetpotato [9]. These shortcomings limit their application in sweetpotato, and the dosage must be optimized before their use on different cultivars.

Melatonin (MT), an indole alkaloid compound, was first discovered in the pineal gland of cattle brain and is widely distributed in various organisms [12]. MT is regarded as a new plant hormone and also a kind of broad-spectrum and effective natural antioxidant [13], which is involved in many biological processes in plants such as root development, leaf senescence, flowering and vegetative growth, fruit maturation, circadian rhythm, photoprotection, seed germination, and various stress responses [14]. Notably, MT has been proven to have positive effects on the retention of storage quality of agricultural products, including grape (Vitis vinifera L.), cabbage (Brassica oleracea var. capitata Linnaeus), peach (Prunus persica Batsch) and broccoli (Brassica oleracea L. var. italic) [15,16,17,18]. However, published studies have mainly focused on above-ground organs, including fruits, leaves and sprouts, and flowers, and no studies is conducted to investigate the effects of MT on the storage quality of underground organs such as sweetpotato SRs and potato tubers.

In this study, ‘Kokei 14’, a sweetpotato cultivar for table use, was employed to investigate possible roles of MT in postharvest storage of sweetpotato SRs under cold storage. After curing, SRs were stored at 15 °C (control) and 15 °C in combination with the application of exogenous MT. Then, the effects of MT on sprouting, weight loss, respiration, lignification, sugar metabolism, hormone content, and antioxidant enzyme were investigated. The possible mechanisms underlying the regulation of MT with respect to the postharvest quality of sweetpotato SRs were proposed. This study provides a new method for postharvest storage of sweetpotato.

2. Results

2.1. Effects of MT on Dry Matter Content, Respiration and Sprouting

During cold storage at 15 °C, the dry matter content of SRs decreased with the extension of storage time (Figure 1A). The exogenous application of MT significantly alleviated the decrease in dry matter content. In addition, the weight loss percentage was also inhibited by MT treatment (Figure 1B). Furthermore, the respiratory rate of SRs was significantly inhibited by MT treatment at 10 and 20 days after storage (DAS) (Figure 1C). The data above suggest that MT could inhibit the weight loss of SRs via inhibiting respiration.

Notably, the sprouting rate of SRs and bud number per SR both increased with the storage time during cold storage at 15 °C (Figure 1D–F). MT treatment significantly inhibited the sprouting rate and the bud number per SRs. The data above indicate that MT treatment improved the storage quality of sweetpotato by inhibiting weight loss, respiration and sprouting.

2.2. Effects of MT on Carbohydrate Content and Lignification

Starch content in SRs was higher than that of soluble sugars, and among soluble sugars, sucrose content was higher than that of glucose and fructose (Figure 2), which is similar to the findings of a previous study [19]. As compared to control (15 °C), MT treatment increased the starch content significantly at 10, 30 and 40 DAS (Figure 2A). During the cold storage at 15 °C, the content of soluble sugars (glucose, fructose and sucrose) dramatically increased at the early stage and subsequently decreased slightly at the late stage (Figure 2B–D). MT treatment elevated the content of soluble sugars significantly, especially at the late stage of storage. In addition, hexose/sucrose ratio was increased by MT treatment at 10 and 20 DAS (Figure 2E). The measurement of lignin content showed that lignin content under cold storage at 15 °C increased with storage period, whereas MT treatment alleviated the increase in lignin content (Figure 2F). Collectively, MT treatment increased the content of starch and soluble sugars but decreased lignin content.

2.3. Effects of MT on the Activities of β-Amylase and PAL

The activity of β-amylase was then measured, which is the major enzyme degrading starch in sweetpotato SRs [20]. The data showed that MT treatment totally showed an inhibitory effect on β-amylase activity and significantly decreased the activity of β-amylase at 20, 30 and 40 DAS (Figure 3A). Thus, MT treatment increased starch content, possibly via inhibiting the activity of β-amylase. Phenylalanine ammonialyase (PAL) is a key enzyme in lignin biosynthesis, which catalyzes the crucial initial step in lignin biosynthesis [21,22]. The measurement on PAL showed that MT treatment inhibited PAL activity during 20–40 DAS (Figure 3B), which was consistent with reduced lignin content from MT treatment.

2.4. Effects of MT on the Activities of Sucrose-Degrading Enzymes

There are two kinds of sucrose-degrading enzymes in higher plants: invertase (INV) and sucrose synthase (Sus). Invertase can be further divided into cell wall invertase (CWIN), vacuolar invertase (VIN), and cytoplasmic invertase (CIN) based on their subcellular localization [23]. The studies show that sucrose-degrading enzymes regulate the content of starch and soluble sugars in crops such as potato (Solanum tuberosum L.), sweetpotato, and carrot (Daucus carota) [24,25,26]. Therefore, activities of sucrose-degrading enzymes were investigated to identify the key sucrose-degrading enzymes in the regulation of MT to sugar content in sweetpotato. During the cold storage at 15 °C, the activities of three kinds of invertases (CWIN, VIN and CIN) all showed a decreasing tendency (Figure 4A–C), whereas the activity of Sus showed an increasing tendency (Figure 4D). Notably, MT treatment had different effects on the activities of INV and Sus. The application of MT decreased the activities of CWIN (10, 30 and 40 DAS), CIN (10–40 DAS) and VIN (10–30 DAS) but increased the activity of Sus (20–40 DAS). These results indicate that Sus may be responsible for the increased hexose content by MT treatment under cold storage.

2.5. Effects of MT on the Gene Expression of Sucrose-Degrading Enzymes

The data above suggest that MT treatment had significant effects on the activities of the sucrose-degrading enzyme. In order to identify the key genes responsible for the changes in enzyme activity, the gene expressions were measured. Based on the previous study in sweetpotato, there are four, six, 12 and nine genes identified from the gene families of CWIN, VIN, CIN and Sus, respectively, which were named IbCWIN1-4, IbVIN1-6, IbCIN1-12 and IbSus1-9 [19,27]. Our preliminary assay showed that only two CWIN genes (IbCWIN1 and IbCWIN2), two VIN genes (IbVIN1 and IbVIN2), four CIN genes (IbCIN3, IbCIN7, IbCIN8 and IbCIN9), and three Sus genes (IbSus5, IbSus7 and IbSus9) are highly expressed in sweetpotato SRs (data not shown).

The qRT-PCR assay showed that MT treatment inhibited the expression of IbCWIN2 (30 and 40 DAS), IbCIN7 (10–40 DAS), IbCIN8 (20 and 30 DAS) and IbVIN1 (10 and 40 DAS) (Figure 5A–D) and had no effects on the expression of the other INV genes highly expressed in SRs Notably, IbCIN7 was the gene most inhibited by MT among all four inhibited INV genes. Different from INV expression, MT treatment enhanced the expression of IbSus5 (20 and 40 DAS) and IbSus9 (30 and 40 DAS) without affecting the expression of IbSus7 (Figure 5E,F). The results above show that, consistent with the findings in enzyme activity, MT treatment inhibited the expression of INV genes but enhanced the expression of Sus genes in SRs.

2.6. Effects of MT on the Activities of Hexose Kinase (HK) and Hormone Content

To elucidate the inhibitory effects of MT on respiration, HK activity was assayed. The data showed that the cold storage under 15 °C decreased HK activity significantly, whereas the application of MT further decreased the activity of HK (Figure 6A). In detail, MT significantly decreased HK activity at 10, 20 and 30 DAS.

As stated above, MT treatment inhibited the sprouting of sweetpotato during cold storage. Therefore, the contents of the spouting-related plant hormones, including gibberellic acid (GA), jasmonic acid (JA) and abscisic acid (ABA) [28,29], were measured. The data showed that under cold storage at 15 °C, GA content remained stable without any big fluctuation during the whole storage period. However, MT treatment led to a dramatic decrease in GA content (Figure 6B). JA content also remained stable during cold storage at 15 °C, whereas MT treatment increased JA content dramatically at 40 DAS (Figure 6C). MT did not alter the change tendency of ABA content but increased the content of ABA (Figure 6D). Collectively, as compared to the cold storage at 15 °C, MT decreased the content of GA but increased the content of ABA and JA.

2.7. Effects of MT on the Activities of Antioxidant Enzymes

During cold storage, the activities of antioxidant enzymes usually increase in various agricultural products to avoid the incidence of chilling injury [30]. Our assay showed that during cold storage at 15 °C, the activities of superoxide dismutase (SOD) and peroxidase (POD) kept increasing, whereas the activity of catalase (CAT) increased first and then decreased quickly (Figure 7A–C). MT treatment did not alter the pattern of change in the activity of antioxidant enzymes but increased their activity under cold storage. In detail, MT treatment increased the activities of SOD and CAT at 20, 30 and 40 d and increased POD activity at 10, 20 and 30 DAS.

3. Discussion

Plenty of studies have revealed that application of exogenous MT improves storage quality of various agricultural products, e.g., sweet cherry (Prunus avium L.) [31], strawberry (Fragaria×ananassas Duch.) [32] and cherry tomato fruit (Solanum lycopersicum var. cerasiforme) [33]. This study showed that MT treatment also improved the postharvest quality of sweetpotato SRs by inhibiting sprouting, weight loss and lignification and simultaneously elevating carbohydrate content and antioxidant capacity under cold storage (Figure 8).

3.1. MT Inhibited Sprouting by Differentially Regulating ABA/JA and GA Content

It has been extensively revealed that MT activates seed germination and subsequent seedling growth of many crops, such as mung bean (Vigna radiata) and soybean (Glycine max) [30,34]. To date, however, no study has been conducted to investigate the effects of MT on the sprouting of tubers or storage roots during postharvest storage. Different from dry seed of many crops, sweetpotato SRs only have a short dormancy period after harvest and are prone to sprouting even under cold storage conditions, which inevitably decreases the marketability of sweetpotato [9]. Our study showed that MT treatment actually inhibited, instead of activated, the sprouting of sweetpotato SRs, as indicated by decreased sprouting rate and bud number per SR. Although our findings in sweetpotato SRs were different from the findings in seeds of many crops, one recent study has also revealed that MT treatment showed inhibitory effects on germination of Arabidopsis seed [35].

Hormones play important roles in seed germination. ABA/JA and GA are well-known as germination-related phytohormones, which inhibit and promote germination and seeding establishment, respectively [30,36,37]. The measurement of endogenous hormones during sprouting of sweetpotato SRs indicates that low ABA concentration is crucial for sprouting, and GAs are not key hormones for sprouting [38]. However, a previous study investigating exogenous application of GA or GA synthesis inhibitor (Paclobutrazol and Prohexadione Calcium) showed that GAs are involved in stimulation of sprouting in sweetpotatoes [39]. Contrasting effects of MT on germination may result from differential effects of MT on germination-related phytohormones, i.e., ABA/JA and GA. In plants, MT treatment usually elevates the content of GA but downregulates the content of ABA and JA [40]. For example, the most recent study in cherry tomato fruits showed that MT treatment decreased the levels of endogenous ABA and JA but increased the levels of GA [33]. However, several contrasting findings have also been revealed. For example, MT elevated, rather than decreased, the content of ABA in leaves of barley (Hordeum vulgare) [41] and Wild ryegrass leaves (Elymus nutans) [42]. A similar phenomenon also exists in the regulation of MT to the content of ethylene (ETH). For example, MT treatment elevated the level of ETH production and promoted postharvest ripening of apple fruits (Malus pumila Mill.) [43] but suppressed ETH production during postharvest storage in cherry tomato fruit [33]. It appears that the outcomes in the interaction of MT with other plant hormones may vary with plant species or organ types.

Our study showed that MT treatment increased the content of ABA and JA but decreased GA content in sweetpotato SRs, which may contribute to the inhibition of MT on the sprouting of sweetpotato SRs. Similarly, the suppression of MT treatment on germination of Arabidopsis seed is attributed to the increased ABA level and upregulated expression of ABA-responsive genes [35]. Notably, our study showed that GA may play positive roles in the sprouting of sweetpotato SRs. In addition, it is well established that JA is involved in the regulation of plant resistance to pathogen infection [44]. Indeed, postharvest melatonin treatment induced disease resistance of tomatoes (Solanum lycopersicum) by activating JA pathway [45]. Thus, potential roles of MT in disease resistance during sweetpotato storage should be explored in future research.

3.2. MT Reduced Weight Loss and Lignification by Inhibiting Respiration

Respiratory metabolism is the normal process of agricultural products after harvest, which consumes carbohydrates and other compounds [46]. A high respiration rate contributes to the postharvest senescence and rapid deterioration of horticultural products, including weight loss, lignification, and expenditure of nutritional compounds such as starch and soluble sugars [47,48]. There is a direct and positive relationship between the storage time of agricultural products and respiration rate [49]. Hexokinase (HK), catalyzing hexose phosphorylation, makes a pivotal contribution to sugar metabolism and energy production via facilitating respiration [50]. Ectopic overexpression of Arabidopsis hexokinase in tomato plants induced rapid senescence [51]. Therefore, the inhibition of respiration can delay the senescence of agricultural products during storage.

Cold storage at 13–16 °C is the most used method to store sweetpotato SRs since it can effectively maintain the quality of sweetpotato by inhibiting respiration and senescence [10]. MT has been proven to reduce respiratory rate in sweet cherry, strawberries and pears (Pyrus communis L.) [31,32,52] and thus extends the storage duration of agricultural products. Our assays showed that, as compared to cold storage, MT application further inhibited respiration rate of sweetpotato SRs. Consistent with the reduced respiration rate, HK activity of sweetpotato SRs was also reduced by MT treatment, indicating MT might decrease respiration by inhibiting HK activity. Consistently, MT treatment decreased weight loss percentage (DW) and increased dry matter content of sweetpotato SRs, indicating reduced weight loss from MT treatment.

Furthermore, MT treatment reduced lignin content of sweetpotato SRs, which is consistent with findings during cold storage of many horticultural products. For example, MT treatment during cold storage decreased lignin content of green asparagus (Asparagus officinalis L.), bamboo shoot (Phyllostachys edulis), and loquat fruit [Eriobotrya japonica (Thunb.) Lindl.] [48,53,54]. Simultaneously, the activity of PAL, a pivotal enzyme in the biosynthesis of lignin, was also significantly inhibited by MT treatment in sweetpotato SRs. The data above show that MT delayed the lignification of sweetpotato under cold storage. However, this finding is not consistent across species. Many studies under ambient temperature showed that melatonin was found to promote lignin biosynthesis. For example, when stored at room temperature (22 ± 1 °C), MT increased the resistance of cherry tomato fruits to gray mold by facilitating lignin biosynthesis [55]. MT treatment during the growth season of herbaceous peony (Paeonia lactiflora Pall.) in an open field increased stem strength by increasing lignin content [56]. MT treatment facilitates the wound healing of potato after harvest by increasing the deposition of lignin and suberin polyphenolic [57]. It appears that the effects of melatonin on lignin biosynthesis are temperature-dependent [56]. A molecular study still needs to be conducted in order to fully elucidate the mechanisms underlying the differential regulation of MT to lignification.

3.3. MT Increased Carbohydrate Content by Regulating Sucrose and Starch Metabolism

Carbohydrates (starch and soluble sugar) are the major components of sweetpotato SR, which accounts for 60–70% of its dry matter [6]. During cold storage of agricultural products, the degradation of starch and sucrose usually accelerates to produce a high content of hexose (mainly glucose and fructose), which not only alleviates chilling injury but also increases the sweetness of products [58,59]. In addition, soluble sugars also serve as a substrate of respiratory metabolism, and therefore affect the shelf life of agricultural products [10]. It has been reported that cold storage induces the accumulation of soluble sugars in potato and sweetpotato, which is referred to as cold-induced sweetening (CIS) [19,24]. MT treatment under cold storage facilitates the accumulation of soluble sugars in various agricultural products such as kiwiberry fruit (Actinidia arguta), pear fruit and flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee) [60,61,62]. Our assay showed that MT treatment not only increased the content of soluble sugars (glucose, fructose and sucrose) but also increased starch content in sweetpotato SRs.

In plants, INV and Sus degrade sucrose into glucose and fructose, therefore affecting the content and composition of soluble sugars [63]. It has been revealed that MT treatment usually represses the activities of INV and/or Sus. In apple fruits, MT treatment inhibited the activities of CWIN, VIN, CIN and Sus [46]. MT treatment reduced CIN activity in pear fruits [61] and improved the storage quality of flowering Chinese cabbage by suppressing the activities of CWIN, VIN, and CIN [62]. In addition, MT inhibited the expression of Sus in SRs of cassava (Manihot esculenta Crantz) during storage [64]. However, contrasting results were also reported in kiwiberry fruits, in which MT application upregulated the activities and expression of CIN and Sus but downregulated those of CWIN and VIN [60].

Our study showed that, accompanied by an increase in soluble sugar content, MT treatment decreased the activities of CWIN, CIN, and VIN but increased the activity of Sus in sweetpotato SRs, which were verified by the changed expression levels of sucrose-degrading enzyme genes. The results can effectively explain the increased sucrose content by MT treatment since the activities of most sucrose-degrading enzymes were inhibited. However, they cannot explain why the content of glucose and fructose was also increased in sweetpotato SRs by MT treatment. In postharvest storage of apple fruits, MT treatment increased the content of soluble sugars via inhibiting respiration but not via increasing sucrose-degrading capacity since MT decreased the activity of CWIN, VIN and CIN [46]. In this study, respiration and starch degradation were further investigated since they also exert important roles in the homeostasis of soluble sugar levels, which affect soluble sugar content negatively and positively, respectively. As discussed above, MT treatment inhibited the respiration rate and HK activities of sweetpotato SRs, which may increase hexose and sucrose content by reducing their expenditure via glycolysis.

VIN is the key sucrose-degrading enzyme in cold-induced sweetening (CIS) of potato and sweetpotato [19,24]. However, our assay indicated that MT treatment inhibited the activity of INV, including VIN, but increased the activity of Sus. Sus is a glycosyl transferase, which reversibly converts sucrose in the presence of UDP into UDP-glucose and fructose [23]. UDP-glucose can act as the substrate for the biosynthesis of starch [63], and fructose contributes to the accumulation of hexose. Actually, a recent study has revealed that Sus is also responsible for the cold-induced sweetening in cold-stored potato tubers [65]. Thus, increased content of soluble sugar in sweetpotato SRs should be attributed to the combined effect of reduced respiration rate and induced Sus activity by MT treatment.

Notably, our assay showed that MT treatment not only increased the content of soluble sugars but also increased starch content in sweetpotato SRs. On one hand, this may result from the decreased β-amylase activity by MT treatment. On the other hand, increased Sus activity and sucrose content by MT treatment may also contribute to the increased starch content since Sus facilitates the biosynthesis of starch by providing UDP-glucose as substrate, as reported in potato tubers [66,67].

3.4. MT Improved Storage Quality of Sweetpotato via Elevating Antioxidant Capacity

Sweetpotato originates from tropical regions, which makes it vulnerable to chilling injury during cold storage, such as surface pitting, decreased dry matter content, and increased susceptibility to decay during storage [68]. It has been well established that chilling injury of cold-sensitive plants results from reactive oxygen species (ROS) burst, which causes lipid peroxidation and protein oxidation [69]. In addition, ROS also promotes senescence and lignification of agricultural products during cold storage [48]. In sweetpotato, antioxidant capacity is tightly related to cold tolerance and storage quality of sweetpotato [69].

MT is not only a kind of natural antioxidant but also an activator of antioxidant systems in plants and thus can alleviate chilling injury of agricultural products by scavenging ROS during cold storage [13,30]. For example, MT increased the antioxidant capacity of soybean seedlings by increasing the content of isoflavone and the activity of SOD and CAT [34]. In sweetpotato, exogenous MT alleviates the browning of fresh-cut sweetpotato by increasing both enzymatic (SOD, POD and CAT) and non-enzymatic (ascorbic acid and glutathione) antioxidant ability [14]. However, no study has been conducted to investigate the effects of MT on the activities of antioxidant enzymes during cold storage of intact sweetpotato SRs. Consistent with findings listed above, our assays showed that MT treatment significantly increased the activities of SOD, POD and CAT. However, no chilling injury was found in either control or MT treatment. Therefore, it was postulated that increased antioxidant capacity by MT treatment may improve storage quality of sweetpotato SRs via inhibiting lignification and senescence, as reported in green asparagus (Asparagus officinalis L.) [48].

4. Materials and Methods

4.1. Materials

Sweetpotato cultivar ‘Kokei 14’ was cultivated at the Batou Experimental Base of Hainan University (18.38° N, 109.15° E) in Sanya, China, and harvested at 120 d after planting. SR weighing 100–120 g was employed to investigate possible roles of MT in postharvest storage of sweetpotato SRs. Solutions with 0.25, 0.5, 0.75, 1 and 1.25 mM of MT (S20287, Orileaf, Shanghai, China) were first used in the preliminary experiment to find the optimal MT concentration for the postharvest treatment of sweetpotato SRs. The 0.5 mM MT solution showed the best effects in the retention of storage quality of sweetpotato and thus was used in the following formal experiment. In detail, after curing, sweetpotato SRs in control group were sprayed with distilled water, and those in MT treatment group were sprayed with 0.50 mM of MT solution. After drying at room temperature for two hours, SRs in the control and MT group were stored separately at a temperature of 15 °C and a relative humidity of 85%. Each group included four replicates, and each replicate had forty sweetpotato SRs. The sampling of sweetpotato flesh was conducted at 0, 10, 20, 30 and 40 DAS, and then the samples were stored at −80 °C for further assays.

4.2. Dry Matter Content, Weight Loss Percentage, Sprouting Rate and Bud Number per SR

For the measurement of dry matter content, the sweetpotato SRs were first weighed, and then SRs were cut into pieces and dried in an oven at 80 °C for 4 d until they reached a constant weight. After weighing the dry weight, dry matter content of SRs was then calculated according to the following formula: dry matter content = (fresh weight/dry weight) × 100%.

For the calculation of weight loss percentage, the fresh weight of the same six SRs was weighed at each timepoint (i.e., 0, 10, 20, 30 and 40 DAS), and then the corresponding dry weight was calculated according to the dry matter content measured above. Finally, weight loss percentage was calculated according to the following formula: weight loss percentage = (dry weight at 0 DAS —dry weight at each timepoint)/dry weight at 0 DAS × 100%.

For sprouting rate and bud number per SR, forty SRs were observed at each timepoint. The number of SRs with visible sprouts and the sprout number in each SR were recorded at each time point, and then the sprouting rate and bud number per SR were calculated.

4.3. Measurement of Respiration Rate

The measurement of respiration rate was conducted according to the published method [70]. In brief, six SRs were put into a gas-tight box (7.5 L) after being weighed. An empty box was used as the reference. After incubation in the boxes for 1 h at 25 °C, O_2_ and CO_2_ levels in two boxes were measured using a portable O_2_/CO_2_ headspace analyzer DK190 (Saicheng Electronic Technology Inc., Jinan, China). The respiration rate was expressed as CO_2_ mg·kg^−1^·h^−1^.

4.4. Assay of Content of Carbohydrate and Lignin

Glucose, fructose and sucrose were quantified spectrophotometrically as described by King et al. [71] with some minor modifications. A 0.2 g measure of sweetpotato SR flesh was ground into a fine powder in liquid nitrogen. The powder was extracted with 600 µL of methanol for 15 min at 70 °C. After cooling to room temperature, the samples were added to 400 µL of chloroform and incubated at 37 °C for 5 min. The total 1 mL of extraction mixture was combined with 800 µL of water and shaken vigorously. After centrifugation at 12,000× g for 5 min, the aqueous phase was collected for sucrose, glucose and fructose content assays. Glucose was measured by adding 30 µL of extract into a 0.6 mL glucose assay buffer containing 60 mM Hepes-KOH (pH 7.4), 4 mM MgSO_4_, 3.3 mM ATP, 1 mM NADP and 0.4 U glucose-6-phosphate dehydrogenase and 0.4 U hexose kinase. After a 30 min incubation at 30 °C, the absorbance (A1) was measured at 340 nm. For the fructose assay, 30 µL of extract and 0.8 U phosphoglucose isomerase were added to the glucose assay buffer. After a 60 min incubation at 30 °C, the absorbance (A2) was measured at 340 nm. To assay sucrose, 30 µL of extract, 0.8 U phosphoglucose isomerase and 4 U INV were added to the glucose assay buffer. After a 60 min incubation at 30 °C, the absorbance (A3) was measured at 340 nm. The quantity of glucose (A1), fructose (A2-A1) and sucrose (A3-A2) was calculated based on the molar extinction coefficient of NADPH.

Starch content was measured according to Smith and Zeeman [72]. A 0.2 g measure of the flesh of sweetpotato SR was homogenized in a mortar in 0.75 mL of 80% ethanol with a pestle and transferred to a 1.5 mL Eppendorf tube. The mortar and pestle were washed with another 0.75 mL of 80% ethanol, which was combined with the previous homogenate. The resultant 1.5 mL of homogenate was centrifuged at 12,000× g for 5 min. After discarding the supernatant, the pellet was washed twice with 1 mL of 80% ethanol and then dried at room temperature for 15 min. The pellet was resuspended in 0.6 mL of H_2_O and heated at 100 °C for 15 min. After neutralizing with 0.6 mL of 0.2 M acetate buffer (pH 5.5), 1 U α-amylase and 6 U α-amyloglucosidase were added to the tube prior to 4 h of incubation at 37 °C. After centrifuging at 10,000× g for 5 min, the derived glucose from starch degradation in the supernatant was measured as described above.

The lignin content was determined using the method of Boonsiriwit et al. [48]. 1 g flesh of sweetpotato SR was homogenized with 5 mL of 95% ethanol (v/v) using a pestle and mortar and then centrifuged at 10,000× g for 10 min at 4 °C. The residue was washed three times with 3 mL of ethanol:hexane (1:2, v/v) solution. The residue was dissolved in 1 mL of acetyl bromide:acetic acid (1:3, v/v) solution and allowed to digest in a 50 °C water bath for 2 h with shaking every 30 min. After cooling and centrifugation at 3000× g for 15 min, the digested solution (0.5 mL) was added to a tube containing 6.5 mL of acetic acid and 2.0 mL of 0.3 M NaOH. The contents were mixed, and hydroxylamine hydrochloride solution (1.0 mL) was added, followed by additional mixing of the contents, after which the absorbance at 280 nm was measured.

4.5. Assay of Activities of INV, Sus and HK

Activities of CWIN, VIN, CIN, Sus, and hexose kinases were assayed spectrophotometrically according to Tomlinson et al. [73]. A 0.2 g measure of the flesh of sweetpotato SR was ground into fine powder in liquid nitrogen. The powder was homogenized in 0.6 mL 100 mM Hepes-KOH extraction buffer (pH 7.4), which contained 5 mM MgCl_2_, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulphonyl-fluoride, 5 mM diothiothreitol (DTT), 1 mL L^−1^ Triton X-100, 200 mL L^−1^ glycerol and 5 mM thiourea. The mortar and pestle were washed with 0.2 mL extraction buffer. The extract was centrifuged at 14,000× g at 4 °C for 10 min. The supernatant was collected for the assays of VIN and CIN activities. The pellet was resuspended in 1 mL of extraction buffer and then centrifuged as described above. After discarding the supernatant, the pellet was resuspended in 0.8 mL extraction buffer for the assay of CWIN activity.

For the assay of CWIN activity, 40 µL of the pellet fraction was added to 360 µL of 50 mM sodium acetate buffer (pH 4.7) containing 0.1 M sucrose. For the assay of VIN activity, 40 µL of the supernatant fraction was added to 360 µL of 50 mM sodium acetate buffer (pH 4.3) containing 0.1 M sucrose. For the assay of CIN activity, 40 µL of the supernatant fraction was added to 360 µL of 50 mM N, N-Bis (2-hydroxyethyl) glycine (Bicine)-KOH buffer (pH 7.6) containing 0.1 M sucrose. At time zero, these three enzyme assay mixes were transferred to 30 °C for 1 h for reaction, then to 85 °C for 3 min to stop the reaction. Controls were included, in which the incubation at 30 °C was omitted. Prior to heating at 85 °C, the assays and controls for acid invertases (CWIN and VIN) were alkalinized by the addition of 60 µL 1 M Tris-HCl (pH 8.0). To remove possible pigment and debris, the enzyme assay mix was added to the same volume of chloroform and then centrifuged at 14,000× g for 5 min. The supernatant was collected for glucose assays. The 140 µL supernatant was added to a 380 µL glucose assay mix, which contained 100 mM Hepes-KOH (pH 7.4), 2.25 mM MgCl_2_, 1.1 mM ATP, 1.1 mM NADP, 0.4 U HK and 0.4 U glucose-6-phosphate dehydrogenase. After incubation at 30 °C for 30 min, the production of NADPH from the reaction was measured spectrophotometrically from the increase in absorbance at 340 nm. Glucose released from sucrose hydrolysis by CWIN, VIN or CIN was quantified based on the molar extinction coefficient of NADPH (6.22 mM^−1^ cm^−1^).

For the assay of Sus activity, 20 µL of the supernatant fraction and 20 µL of the pellet fraction were added to 360 µL of 100 mM Hepes-KOH (pH 7.0) containing 4 mM UDP and 0.2 M sucrose. UDP was omitted in the control. At time zero, all assays were transferred to 30 °C for 30 min. Reactions were stopped by transferring to 85 °C for 3 min. The same volume of chloroform was added to the enzyme assay mix. After centrifugation at 14,000× g for 5 min, the supernatant was collected for glucose assays. The glucose assay was identical to that for INV, except for the addition of 0.8 U phosphoglucose isomerase to the glucose assay mix to convert fructose 6-phosphate to glucose 6-phosphate.

For the hexose kinases assay, 20 µL of the supernatant fraction and 20 µL of the pellet fraction were incubated at 30 °C for 20 min in a final volume of 360 µL buffer containing 50 mM Hepes-KOH (pH 8.0), 2.5 mM MgCl_2_, 1.5 mM ATP, and 5 mM glucose. Controls did not contain ATP and glucose. After heating at 85 °C for 3 min, assays of glucose 6-phosphate produced by hexose kinases were conducted as detailed above, except that they lacked ATP and HK.

4.6. Assay of Activity of Antioxidant Enzymes, PAL and β-Amylase

The activities of antioxidant enzymes, including SOD, POD, and CAT, were assayed based on the method of Lee and Lee [74]. A 0.5 g of the flesh of sweetpotato SR was homogenized in 100 mM potassium phosphate buffer (pH 7.5) containing 0.5 mM EDTA and 1 mM ascorbate acid, 1% PVP (w/v) at 4 °C. The homogenate was centrifuged at 12,000× g for 15 min at 4 °C. The resultant supernatant was collected. A 30 µL supernatant was added to 1.5 mL of 50 mM potassium phosphate buffer (pH 7.8) containing 13 mM methionine, 63 mM NBT and 1.3 mM riboflavin. The well-mixed solution was then exposed to light (100 µmol m^−2^ s^−1^) for 15 min, and then the absorbance at 560 nm was measured. POD activity was assayed by monitoring the oxidation of ascorbic acid at 290 nm. A 100 µL supernatant was added to 1 mL 50 mM potassium phosphate buffer (pH 7.0) containing 0.5 mM ascorbate acid, 0.1 mM EDTA and 1.5 mM H_2_O_2_. The absorbance decrease at 290 nm was recorded at 25 °C in 30 s, and then POD activity was calculated using the extinction coefficient of ascorbate acid (2.8 mM^−1^ cm^−1^). CAT activity was assayed by monitoring the degradation of H_2_O_2_ by CAT at 240 nm. 200 µL supernatant was added to 1 mL of 25 mM potassium phosphate buffer (pH 7.0) containing 10 mM H_2_O_2_ and 0.1 mM EDTA, and the absorbance decrease at 240 nm was recorded at 25 °C in 45 s. CAT activity was calculated using the extinction coefficient of H_2_O_2_ (39.4 mM^−1^ cm^−1^).

The activity of PAL was measured using the method of Boonsiriwit et al. [48]. A 0.5 g measure of the flesh of sweetpotato SR was ground with 6 mL of 50 mM Tris-HCl buffer (pH 8.8) containing 15 mM β-mercaptoethanol, 5 mM EDTA, 5 mM ascorbic acid, and 0.15% (w/v) polyvinylpyrrolidone (PVP). The homogenate was centrifuged at 12,000× g for 30 min at 4 °C. A 0.5 mL measure of supernatant was added to the reaction mixture containing 16 mM L-phenylalanine, 50 mM Tris-HCl buffer (pH 8.9), 3.6 mM NaCl. After incubation at 37 °C for 1 h, the reaction was stopped by adding 500 µL of 6 M HCl. PAL activity was determined by measuring absorbance at 290 nm.

The activity of β-amylase was determined using a testing kit (BC2045, Solarbio, Beijing, China). A 0.1 g measure of the flesh of sweetpotato SR was homogenized in 0.8 mL distilled water. The homogenate was then placed at room temperature for 15 min with shaking every 5 min. After centrifuging at 6000× g for 10 min at 25 °C, the supernatant was collected and diluted into 10 mL for the assay of β-amylase activity. A 75 µL measure of diluted supernatant was added to two 2 mL EP tubes. One tube was boiled for 5 min and used as the control tube, and the other tube was used as the testing tube, which was added to 75 µL Reagent B and incubated in a water bath at 70 °C for 15 min. After cooling to root temperature, the control tube was added to 150 µL Reagent A and 75 µL Reagent B, and the testing tube was added to 150 µL Reagent A. After incubation at 40 °C for 5 min, the tubes were boiled for 10 min. The cooled solution was taken out and β-amylase activity was determined by measuring absorbance at 540 nm.

4.7. qRT-PCR Assay of Genes of Sucrose-Degrading Enzymes

Total RNA was purified using the RNAprep Pure Polysaccharide & Polyphenol Plant Kit (DP441, Tiangen Inc., Beijing, China) according to the manufacturer’s instructions. cDNA was synthesized using HiScript Ⅲ All-in-one RT SuperMix Perfect for qPCR (R333-01, Vazyme Inc, Nanjing, China). qRT-PCR was conducted using SYBR Green-based kit (AQ601-02, TransGen Biotech Inc., Beijing, China) with IbActin as the reference gene. The primer pairs of interesting genes were designed using the Primer-BLAST on NCBI website (Table 1). Relative expression levels of interest genes were calculated using the 2^−ΔΔCT^ method.

4.8. Measurement of Plant Hormones

The levels of GA, ABA and JA were measured according to the published method with minor modifications [75]. In brief, 0.2 g sweetpotato flesh was homogenized and extracted in 1 mL of methanol: acetic acid: water (12:3:5 [v/v/v]) for 12 h at 4 °C. After centrifugation for 10 min at 8000× g (4 °C), the supernatant was used to determine plant hormone content, and the pellet was extracted at least three times. The extraction solution was concentrated with a vacuum rotary evaporator. The residue was dissolved with 0.5 mL methanol: acetic acid: water (12:3:5 [v/v/v]) and filtered through a 0.22 μm filter prepared for HPLC (Agilent Technologies 1200 series, Santa Clara, CA, USA) analysis. The flow rate was 0.8 mL min^−1^, and the injection volume was 10 μL. The quantities of plant hormones in the sample were expressed on a fresh weight basis (μg·g^−1^ FW).

4.9. Statistical Analyses

In all experiments, each group included four biological replicates. Data are presented as mean ± standard error (SE). A t-test was carried out for difference analyses between control and MT treatment at the same timepoint of storage using IBM SPSS Statistics 27 (SPSS Inc., Chicago, IL, USA). All figures were completed using GraphPad Prism 9.5 (Dotmatics, Boston, MA, USA).

5. Conclusions

This study showed that MT treatment improved postharvest quality of sweetpotato under cold storage through inhibiting sprouting, respiration, weight loss, lignification and simultaneously elevated carbohydrate content and antioxidant capacity under cold storage. Furthermore, possible mechanisms were proposed. Notably, contrasting findings of MT treatment on lignification, sprouting and the content of GA and ABA were found among different agricultural products, including sweetpotato, and the possible underlying mechanisms should be further investigated in the future. The potential roles of MT in disease resistance during sweetpotato storage remain to be examined since MT treatment enhanced the level of JA, a well-known disease-related hormone.