Introduction

Cold storage is a useful method to delay decay and to maintain the quality of postharvest fruit. However, peach (Prunus persica) is susceptible to chilling injury (CI). Classic visual symptoms of CI in peach fruit are internal browning and flesh mealiness. These symptoms limit postharvest life and reduce consumer acceptability (Lurie and Crisosto 2005). Consequently, understanding of the mechanisms of mitigation of CI by potential technologies in fruit is imperative.

Many technologies have been employed to reduce CI in fruit. Heat treatment has been shown to modulate soluble sugar metabolism and induce chilling tolerance in loquat fruit (Shao et al. 2013). Brassinolide (Aghdam and Mohammadkhani 2014), methyl jasmonate (MeJA) (Jin et al. 2014), and gibberellic acid (GA3) (Zhu et al. 2016) applications have been found to effectively enhance chilling tolerance in tomato fruit. A previous study by our group verified that application of an exogenous nitric oxide (NO) donor, sodium nitroprusside (SNP), reduced the levels of lipoxygenase and phospholipase D and activated antioxidant enzymes and small ubiquitin-like modifier (SUMO) in peach fruit, thereby reducing the CI index (Jiao et al. 2019). The mitigation of CI in fruit requires the involvement of endogenous signal compounds (Jiao et al. 2019). It is of great importance to explore the possible downstream signal molecules that mediate NO-mitigated CI in peach fruit. Glycogen synthase kinase-3 (GSK-3), a serine/threonine kinase, has been shown to be involved in many physiological responses in plants under exogenous stimuli, such as wound (Jonak et al. 2000) and salt stress (Chen et al. 2003). Especially, another previous work from our group has reported that GSK-3 mediated NO-induced isoflavone synthesis in soybean sprouts after UV-B treatment (Jiao et al. 2017). However, there are no reports concerning the mediation of NO-alleviated CI by GSK-3 in peach fruit.

Membrane damage in fruit caused by low temperatures leads to elevated malondialdehyde (MDA) contents and electrolyte leakage, which are qualitative indexes of CI (Dong et al. 2012). It has been reported that NO can reduce MDA content and electrolyte leakage and thereby alleviate membrane damage in loquat fruit (Xua et al. 2012) and mango fruit (Ruan et al. 2015). Furthermore, GSK-3 activation and declines of MDA content and electrolyte leakage are the results of defense responses. However, no reports concern the GSK-3-mediated decreases in MDA content and electrolyte leakage under NO treatment in peach fruit.

Alternative oxidase (AOX), a non-energy-conserving terminal oxidase in the plant mitochondrial electron transport chain, plays important roles in metabolic and signaling homeostasis under stressful conditions. It has been shown to be involved in the alleviation of CI induced by methyl jasmonate (MeJA) and methyl salicylate (MeSA) treatments in tomato and sweet pepper (Wang et al. 2005). Additionally, AOX activity was enhanced by NO in tobacco plants (Luisa et al. 2006). No reports are available about the enhancement of AOX by NO in peach fruit. Furthermore, the mechanism by which GSK-3 mediates AOX activation under NO treatment remains to be revealed.

Heat shock proteins (HSPs), a group of conserved proteins, play central roles in maintaining cellular homeostasis (Carranco et al. 1997). Salicylic acid (Wang et al. 2006) and MeJA and MeSA (Ding et al. 2001) applications induce HSPs, thereby mitigating CI in peach and tomato fruit, respectively. No reports are about the induction of HSP by NO in peach fruit. Furthermore, the mediation of HSP activation by GSK-3 under NO treatment in peach fruit remains to be explored.

Plant-specific WRKYs, comprising a family of transcription factors, have been shown to play important roles in various stress responses. WRKY has been shown to be involved in the enhancement of chilling tolerance due to abscisic acid (ABA) treatment in banana fruit (Luo et al. 2017) and to be upregulated by SNP treatment in Larix olgensis Henry (Hu et al. 2015). However, no reports have been conducted on the induction of WRKY by NO and its role in CI alleviation in peach fruit. Serine/threonine kinases, including GSK-3 and mitogen-activated protein kinase (MAPK), have been confirmed to be involved in some biological processes. A WRKY transcription factor was reported to be phosphorylated by MAPK in Arabidopsis (Mao et al. 2011). No reports are available regarding the stimulation of WRKY by GSK-3 in response to NO treatment in peach fruit. Additionally, C-repeat binding factors (CBFs), as another transcription factor, have been verified to play important roles in plant response to cold stress (Oakenfull et al. 2013). Gibberellic acid treatment has been shown to induce gene expression of CBF, a vital regulator in the cold response in tomato fruit (Zhu et al. 2016), and endogenous NO production under cold stress was found to modulate CBF in tomato via nitrosylation (Sehrawat et al. 2013). However, no reports are about CBF induction by GSK-3 in response to NO treatment in peach fruit.

Based on the above observations, we hypothesized that GSK-3 can mediate NO-alleviated CI in peach fruit. The aims of the present study were to investigate the modulation of the CI index, the reductions of MDA content and electrolyte leakage, and the activation of AOX, HSP, WRKY, and CBF by GSK-3 in peach fruit under NO treatment and to reveal the underlying mechanisms.

Materials and Methods

Fruit Material and Postharvest Treatments

Peach fruit (Prunus persica Batsch ‘Jinqiuhongmi’) were harvested at commercial maturity at a local orchard in Beijing, China. Peaches of uniform size and maturity with absence of mechanical damage were chosen and randomly divided into three groups:

(1) Control group (CK): The fruits were immersed in sterile deionized water.

(2) SNP (NO donor): The fruits were immersed in 15 μmol L−1 SNP.

(3) SNP+bikinin (GSK-3 inhibitor): The fruits were immersed in 15 μmol L−1 SNP plus 10 μmol L−1 bikinin.

The fruits in each group were treated for approximately 10 min and then air-dried for 40 min at room temperature. Subsequently, the fruits in all three groups were stored for 28 days at 4 °C and 80% relative humidity. During the 28 days of storage, twenty fruits from each group were collected at 7-day intervals for assays. Each assay was repeated three times. The samples were stored at − 80 °C for determination.

CI Index Detection

The CI index was calculated according to internal browning symptoms. The severity of browning was assessed in a four-stage scale: 0 = no damage, 1 = superficial damage (< 5% damage), 2 = moderate damage (6–25% damage), 3 = severe damage (26–50% damage), and 4 = very severe damage (> 50% damage). The CI index was obtained using the following formula: CI index =Σ [(CI scale) × (number of fruit at that CI)] / [5 × (total number of fruit in each sample)] × 100%.

MDA Content Determination

A total of 1.0 g of peach fruit was ground with 5 mL of 10% trichloroacetic acid and then centrifuged at 12,000×g for 20 min at 4 °C. MDA content was determined using the thiobarbituric acid (TBA) method according to Ding et al. (2016).

Electrolyte Leakage Detection

Thirty disks (5 mm in diameter) were obtained using a cork borer from the skin tissue of 10 fruits. The disks were immersed in distilled water for 10 min and then dried on filter paper. Then, the disks were immersed in 0.3 mol L−1 mannitol for 3 h in a 50-mL plastic centrifuge tube. Electrolyte leakage was determined according to Ding et al. (2015).

AOX Activity Determination

A total of 0.5 g of peach fruit was ground in 10 mL of 10 mmol L−1 phosphate buffer (pH 7.2) at 4 °C. The peach samples were centrifuged at 10,000g at 4 °C for 15 min. Then, AOX activity was assayed using an assay kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China).

Gene Expression

Total RNA from each peach sample was collected using an E.Z.N.A.™ Plant RNA Kit (Omega, Norcross, GA, USA; R6827-01) according to the manufacturer’s protocol. Subsequently, synthesis of first-strand cDNA was carried out as described by Zhu et al. (2016). The primers used for quantitative real-time PCR (QRT-PCR) determination are listed in Table 1. The QRT-PCR cycling conditions were as follows: 1 cycle of 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s, and then 63 °C for 25 s. Each analysis was performed with 3 independent biological replicate experiments. For each experiment, 3 technical replicates were carried out.

Table 1. The primers used for qRT-PCR.
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Statistical Analyses

All the data were expressed as the mean ± standard deviation (SD). SPSS 21.0 software (SPSS Inc., Chicago, IL, USA) was used to test for significant differences using the ANOVA procedure and Duncan’s test.

Results

Effects of Exogenous SNP and Bikinin on the CI Index in Peach Fruit

The peach fruit developed CI symptoms after 7 days of cold storage. The CI index gradually increased over the storage period. The development of CI symptoms was delayed in peach fruit treated with exogenous SNP. After 28 days of cold storage, the CI index in peach fruit subjected to SNP treatment was 43%, which represented a 38% decrease relative to the value in the control fruit. More interestingly, the reduction of the CI index by SNP treatment in peach fruit was weakened by bikinin (GSK-3 inhibitor) treatment. After 28 days of storage, the CI index in peach fruit under SNP-plus-bikinin treatment was 55%, representing a 29% increase and 20% decrease relative to the value observed under SNP treatment and control treatment, respectively (Fig. 1).

Fig. 1
figure 1

Effects of SNP and bikinin on the CI index in peach fruit. Values are the mean ± SD (error bars). Values not sharing the same letter are significantly different at p < 0.05

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Effects of Exogenous SNP and Bikinin on Gene Expression of GSK-3 in Peach Fruit

During cold storage, the gene expression of GSK-3 first increased and then decreased under control and exogenous SNP treatment. Exogenous SNP induced the expression of GSK-3. GSK-3 expression under SNP treatment reached a maximum (4.2) at 14 days, with a level 1.8 times the control level. After 28 days of cold storage, GSK-3 expression after SNP treatment was 3.1, which was 1.4 times the control level. Interestingly, the induction of GSK-3 expression by SNP treatment was blocked by bikinin. After 28 days of storage, there was no significant difference in GSK-3 expression between the SNP treatment and the SNP-plus-bikinin treatments (Fig. 2).

Fig. 2
figure 2

Effects of SNP and bikinin on gene expression of GSK-3 in peach fruit. Values are the mean ± SD (error bars). Values not sharing the same letter are significantly different at p < 0.05

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GSK-3-Mediated Prevention of the Increase in MDA Content and Electrolyte Leakage Under SNP Treatment

Over the course of cold storage, MDA content and electrolyte leakage in peach fruit increased continuously under the control and exogenous SNP treatments. The increase in MDA content and electrolyte leakage was prevented by exogenous SNP. After 28 days of cold storage, MDA content and electrolyte leakage were 3.40 mmol/kg FW and 42% under SNP treatment, representing decreases of 35% and 33%, respectively, relative to the control values. Interestingly, bikinin weakened the declines of MDA content and electrolyte leakage induced by SNP treatment. After 28 days of storage, MDA content and electrolyte leakage under SNP-plus-bikinin treatment were 4.21 mmol/kg FW and 52%, representing increases of 23% and 24%, respectively, relative to that under SNP treatment and decreases of 19% and 17%, respectively, relative to the control values (Fig. 3).

Fig. 3
figure 3

Effects of bikinin on MDA content (A) and electrolyte leakage (B) under SNP treatment in peach fruit. Values are the mean ± SD (error bars). Values not sharing the same letter are significantly different at p < 0.05

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GSK-3 Mediation of AOX Activity Under SNP Treatment

Over the course of cold storage, AOX activity first increased and then decreased under the control and exogenous SNP treatments. Exogenous SNP enhanced AOX activity. AOX activity under SNP treatment reached a maximum (60 U/mg FW) at 14 days, with a value 1.7 times the control value. After 28 days of cold storage, AOX activity under SNP treatment was 43 U/mg FW, 1.5 times the control value. Interestingly, the activation of AOX by SNP treatment was blocked by bikinin. After 28 days of storage, AOX activity under the SNP-plus-bikinin treatment was 32 U/mg FW, representing a 26% decrease relative to the activity under SNP treatment, and there was no significant difference in AOX activity between the control and SNP-plus-bikinin treatments (Fig. 4).

Fig. 4
figure 4

Effects of bikinin on AOX activity under SNP treatment in peach fruit. Values are the mean ± SD (error bars). Values not sharing the same letter are significantly different at p < 0.05

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GSK-3-Mediated Induction of Gene Expression of 18.1-kDa class I HSP, WRKY2, and CBF Under SNP Treatment

Over the course of cold storage, the gene expression of 18.1-kDa class I HSP, WRKY2, and CBF in peach fruit first increased and then decreased under the control and exogenous SNP treatments. Exogenous SNP treatment enhanced the gene expression of 18.1-kDa class I HSP, WRKY2, and CBF. The gene expression of 18.1-kDa class I HSP, WRKY2, and CBF under SNP treatment reached maximum values (5, 6.9, and 4.9) at 14 days, 14 days, and 21 days, respectively, with values 1.4, 1.4, and 2.9 times the control values, respectively. After 28 days of storage, there was no significant difference in 18.1-kDa class I HSP expression between the control and SNP treatments, and WRKY2 and CBF expression under SNP treatment was 5.9 and 4.4, respectively, with values 2.0 and 3.7 times the corresponding control levels. More importantly, during storage, bikinin inhibited the gene expression of 18.1-kDa class I HSP, WRKY2, and CBF under SNP treatment. After 28 days of storage, there were no significant differences in 18.1-kDa class I HSP and CBF expression between the SNP and SNP-plus-bikinin treatments, and WRKY2 expression under SNP-plus-bikinin treatment was 4.6, representing a 22% decrease relative to the level under SNP treatment (Fig. 5).

Fig. 5
figure 5

Effects of bikinin on gene expression of 18.1-kDa class I HSP (A), WRKY2 (B), and CBF (C) under SNP treatment in peach fruit. Values are the mean ± SD (error bars). Values not sharing the same letter are significantly different at p < 0.05

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Discussion

Our work showed that during cold storage, SNP treatment significantly reduced the CI index (Fig. 1) in peach fruit, demonstrating that SNP application has potential as a method to mitigate CI in peach fruit.

The elevations of MDA content and electrolyte leakage due to peroxidation and breakdown of unsaturated fatty acids reflect the severity of membrane damage (Ruan et al. 2015). In the present study, SNP treatment effectively prevents both the increase in MDA content and electrolyte leakage in cold-stored peach fruit (Fig. 3). This, in turn, suggests that NO may reduce chilling-induced membrane damages. More interestingly, the mitigation of membrane damage by NO was mediated by GSK-3 (Fig. 3). SNP treatment, as an external signal, did not directly induce defense responses but activated endogenous signaling molecules such as GSK-3 (Fig. 2). Subsequently, the stimulation of the signal compounds modulated the downstream physiological responses (Jian et al. 2005).

Furthermore, AOX activity was increased by SNP treatment (Fig. 4). AOX has been shown to regulate some vital mitochondrial signaling compounds, including superoxide, nitric oxide, and important redox couples (Vanlerberghe 2013). Excess ROS-caused oxidative damage is one of the early responses to CI in fruit (Yao et al. 2018). Apart from the antioxidant enzyme we previously reported (Jiao et al. 2019), AOX also acts as the defense line to scavenge ROS. It has been reported that AOX downregulated superoxide levels, thereby reducing reactive oxygen species (ROS) generation in tobacco leaf (Cvetkovska and Vanlerberghe 2012). Therefore, AOX might enhance chilling tolerance by scavenging ROS. Furthermore, GSK-3 mediated the activation of AOX due to SNP treatment in peach fruit (Fig. 4). Accordingly, AtCBL1 and AtCP1, members of the Ca2+ sensor family called the AtCBL family, can be activated upon the overexpression of AtGSK1 in transgenic Arabidopsis. AtCBL1 is considered to be a Ca2+ sensor involved in stress signal transmission (Kudla et al. 1999), and AtCP1 was identified as a small Ca2+-binding protein with EF hands (Jang et al. 1998). Both of these proteins interact with CBL-interacting protein kinases (CIPKs) (Shi et al. 1999). Hence, GSK-3 acts as an effective mediator of Ca2+ mobilization in plant. Moreover, it has been shown that EGTA (a calcium chelator) weakens the alternative respiratory pathway in chilling-stressed Arabidopsis callus (Leo 2012), indicating that Ca2+ is essential to the induction of the alternative respiratory pathway during cold storage. Thus, Ca2+ might mediate GSK-3-induced AOX activation in peach fruit.

HSP has been identified as another biochemical marker of CI degree in fruit. SNP treatment induced gene expression of 18.1-kDa class I HSP in peach fruit (Fig. 5A). HSP is a member of a group of proteins involved in the responses to stressful conditions that act to protect the plant against damage. Increased HSP expression has been found to be correlated with the enhancement of chilling tolerance (Ding et al. 2001). Moreover, our work suggested that GSK-3 mediated the activation of 18.1-kDa class I HSP stimulated by SNP treatment in peach fruit (Fig. 5A). Accordingly, GSK-3 has been shown to phosphorylate heat shock factor 1 at Ser(303), thereby modulating heat shock factor 1 and HSP expression in mice (Zahra et al. 2010). Therefore, we speculate that NO application induces HSP expression by regulating the phosphorylation of heat shock proteins at some amino acid sites.

Some transcription factors are activated as part of the defense responses to stressful conditions in plants (Cheng et al. 2017). The gene expression of WRKY2 was enhanced by SNP treatment in peach fruit (Fig. 5B). Interestingly, GSK-3 mediated the activation of WRKY2 triggered by SNP treatment in peach fruit (Fig. 5B). It has been reported that the increases in Ca2+ influxes due to injury induce the phosphorylation of WRKY in Arabidopsis thaliana (Yan et al. 2018). Thus, the chilling tolerance enhancement by WRKY possibly involves the activation of Ca2+. Additionally, WRKYs have been shown to regulate a plant immune NADPH oxidase in Nicotiana benthamiana (Adachi et al. 2015). Furthermore, the activities of superoxide dismutase (SOD) and peroxidase (POD), the contents of proline and MDA, and electrolyte leakage have also been shown to be regulated by WRKYs (Yang et al. 2017). Hence, WRKY may alleviate CI through modulating the antioxidant system and alleviating membrane damage. The gene expression of another transcription factor, CBF, was also increased by SNP treatment in peach fruit (Fig. 5C). Accordingly, previous work found that tomato plants overexpressing Arabidopsis CBF1 exhibited greater chilling tolerance than wild-type plants (Hsieh et al. 2004), suggesting that CBF is a key regulator of the cold response. Our work showed that NO could effectively upregulate CBF expression during long-term cold storage in peach, a cold-sensitive fruit. More importantly, our results demonstrated that GSK-3 mediated the enhancement of CBF expression due to SNP treatment in peach fruit (Fig. 5C).

Conclusions

Exogenous SNP treatment protected peach fruit against CI and activated GSK-3. The elevated chilling tolerance in SNP-treated peaches may be due to the decreases in MDA content and electrolyte leakage, resulting in the alleviation of membrane damage. Additionally, the activity of AOX and the gene expression of 18.1-kDa class I HSP, WRKY2, and CBF increased in response to SNP treatment; such increases might activate a positive feedback mechanism and induce chilling tolerance in SNP-treated peaches. However, bikinin, a GSK-3 inhibitor, weakened the SNP-induced effects described above. Overall, GSK-3 mediated the induction of chilling tolerance through reductions in MDA content and electrolyte leakage, activation of AOX, and increases in 18.1-kDa class I HSP, WRKY2, and CBF expression under SNP treatment in postharvest peach fruit. The findings of the current study provide a foundation for in-depth studies of the functional roles of the NO signaling network in fruit during cold storage.