‘Nanguo’ pear (Pyrus ussuriensis Maxim.) is one of the most famous pear varieties in Liaoning Province, China, where it is harvested in September each year. Unfortunately, the fruit ripen and senesce rapidly at room temperature, resulting in pulp softening and browning of the core. Therefore, ‘Nanguo’ pear fruit are commonly refrigerated while being stored. However, long-term cold storage renders the fruit prone to peel browning during shelf life at room temperature, even if the fruit has not ripened or softened (Wang et al., 2017).

Fruit browning after refrigeration signals metabolic disorders owing to low temperature stress, such as an energy deficit because of the cold-induced reduction of ATP production. Reduced ATP production is associated with damage of the mitochondrial structures and inhibition of enzymes involved in mitochondrial function (Atkin and Tjoelker, 2003). Mitochondria are the source of all metabolic energy in plant cells. Maintaining the integrity of mitochondrial structure plays a crucial role in controlling peel browning in postharvest ‘Nanguo’ pear. Wang et al. (2018) found that an intermittent warming during cold storage reduces pericarp browning of ‘Nanguo’ pears after a 120-day storage period, which is associated with modified energy and lipid metabolism. Furthermore, exogenous melatonin treatment retards the increase in cell membrane permeability and maintains a high energy state, thereby delaying the browning of litchi fruit after harvest (Wang et al., 2020). Similarly, Cheng et al. (2015) found that treatment with 1-MCP increases the activity of enzymes related to energy metabolism and alleviates the occurrence of chilling injury in ‘Nanguo’ pears.

The non-protein amino acid, γ-Aminobutyrate (GABA), helps plant cells resist biotic and abiotic stress (Fait et al., 2008). GABA content in plant tissues is relatively low; however, it rapidly increases under abiotic stress, such as chilling, salinity, and drought (Mekonnen et al., 2016; Salvatierra et al., 2016; Li J. X. et al., 2019). Morteza et al. (2016) found that GABA treatment reduces chilling injury in Anthurium cut flowers by increasing the activity of the GABA shunt in order to provide sufficient energy. Reduced cold injury in bananas by GABA is associated with the accumulation of proline and triggers the antioxidant defense system (Wang et al., 2014). GABA immersing can significantly inhibit the browning process of fresh-cut apples during postharvest storage (Gao et al., 2018).

Li J. X. et al. (2019) observed an increase in the endogenous GABA content in ‘Nanguo’ pears treated with GABA. Moreover, GABA treatment enhances the mitochondrial antioxidant response in treated pears, whereby the mitochondrial structure remains intact, and, pericarp browning of the fruit after a 180-day storage is effectively retarded (Li J. X. et al., 2019). GABA is synthesized primarily in plant tissues by the cytoplasmic α-decarboxylation of glutamic acid in an irreversible reaction of the GABA shunt, catalyzed by GABA decarboxylase (GAD), whose activity is regulated by Ca²⁺ and calmodulin (CML). GABA is then transported into the mitochondria, where it is converted to succinic semialdehyde (SSA) by GABA transaminase (GABA-T). In the final step of the synthesis, SSA is reduced by succinate semialdehyde dehydrogenase (SSADH) to form succinic acid, which enters the tricarboxylic acid (TCA) cycle (Guo et al., 2012). Yin et al. (2014) found that CaCl₂ can reduce the harmful effects of soybeans under NaCl stress by changing the contribution ratio of GABA shunt and promoting GABA production.

Ionic calcium (Ca²⁺) is abundantly available in plants and acts as a secondary messenger during plant growth and development. Ca²⁺ plays an important regulatory role in vital processes, such as cell division, polar cell growth, cell differentiation, and apoptosis (Poovaiah and Ready, 1993). Exogenous Ca²⁺ application provides plants the ability to tolerate chilling related stress. Postharvest Ca²⁺ application reduces the severity of chilling injury in fruit flesh by increasing flesh calcium, which in turn delays fruit browning after cold storage (Manganaris et al., 2007). GAD has been identified as a CAM-binding protein in plants, stimulated by Ca²⁺/CAM (Baum et al., 1993; Ling et al., 1994; Kinnersley and Turano, 2000). Ca²⁺-based signals in plants involve a complex mix of different binding proteins that function as Ca²⁺ sensors, such as CAM, CML, Ca²⁺-dependent protein kinase (CDPK), CML B-like protein (CBL), and CBL-interaction kinase (CIPK). These Ca²⁺ sensors constitute a complex signal network in plants enabling specific signal transduction (Batistič and Kudla, 2012). Many previous studies have shown that calcium can promote the accumulation of GABA (Gao et al., 2011; Bai et al., 2013).

In view of the function of GABA in maintaining the integrity of the mitochondrial structure and energy production capacity, and given the relationship between GABA biosynthesis and Ca²⁺, further insight into the protective mechanism mediated by GABA and Ca²⁺ against cold injury in pear fruit will require a thorough study of Ca²⁺, CAM, CML, and GABA contents and distribution in pericarp tissues, as well as GABA shunt-related enzyme activities and gene expression. Here, we studied the effects of exogenous Ca²⁺ treatment on pericarp browning in ‘Nanguo’ pears.

To explore the effect of calcium on the GABA branch, mitochondrial structure, and peel browning, peel browning was observed and the calcium content, mitochondrial microstructure, and the activities and the associated gene expression of a series of GABA-related anabolic enzymes were analyzed. Calcium plays a key role in the selective absorption of ions, signal transmission, and stress resistance in plants. At the cellular level, calcium has important regulatory functions in cell division, polarity formation, growth, differentiation, and apoptosis. It maintains cell wall stability, cell membrane structure, regulates the membrane-bound proteins, and participates in the regulation and control of many physiological and biochemical reactions of plant cells. Various stimuli, such as mechanical stimulation, low temperature, red light, plant hormones, fungal elicitors, hypoxia, and water stress have been shown to affect plant tissues, and the initial reaction almost always causes changes in the intracellular Ca²⁺ concentration (Bush, 1995). Ca²⁺ has been hypothesized to play an important role in plant resistance to stress (Luan et al., 2002). Previous studies have shown that the concentration of Ca²⁺ increases under low temperature stress in both chilling-resistant Arabidopsis spp. and chilling-sensitive tobacco (Nicotiana plumbaginifolia), which presumably triggers the Ca²⁺ signals that precede defense responses to stress (Knight et al., 1996). Further, Ca²⁺ has been shown to positively influence membrane integrity and cell membrane permeability (Picchioni et al., 1996; Picchioni et al., 1998). Ferguson et al. (1985) reported increased absorption of Ca²⁺ by the mitochondria of papaya fruit under low temperature stress. Damage to the cytoplasmic membrane is thought to be one of the most important causes of browning of fruit under cold storage, because damage to the cell membrane can lead to a series of secondary reactions, including the obstruction of energy production in the cytoplasm and the accumulation of toxic compounds (Rui et al., 2010). In the present study, the contents of Ca²⁺ and calmodulin in the pears were increased by Ca²⁺ treatment, which significantly increased the GABA content and effectively promoted the formation and metabolism of the GABA branch.

As a signaling molecule, GABA is widely distributed in prokaryotes and eukaryotes (Breitkreuz et al., 1999), playing an important role in resisting biotic and abiotic stress and participating in osmotic regulation in plants (Kumar et al., 2019). Our previous studies have shown that exogenous GABA treatment increases the endogenous GABA content of refrigerated ‘Nanguo’ pears, reducing ROS content and maintaining enzyme activity and the operation of the mitochondrial oxidative-defense system (Li J. X. et al., 2019). The GABA shunt contains three enzymes, the first of which is GAD, which initiates the decarboxylation of glutamate in the cytosol (Fait et al., 2005). GABA synthesis is terminated in the mitochondria by GABA decomposition into SSA and its conversion to succinic acid, which enters the TCA cycle and produces NADH (Fait et al., 2005; Wang et al., 2019). GABA is converted to succinic acid by the GABA shunt pathway to supplement the disrupted TCA cycle under stress conditions. SSADH is a key enzyme in this metabolic pathway (Bouché and Fromm, 2004). GAD activity is regulated by Ca²⁺ and CML. Many studies have shown that GABA levels increase rapidly in plants to cope with various forms of stress. Further, Bai et al. (2008) and Su et al. (2007) found that promoting GAD activity helps stimulate GABA accumulation. In the present study, Ca²⁺ treatment before storage increased GABA synthesis enzyme-related activities and their relative expression levels, as well as endogenous GABA, Ca²⁺, and CML contents in ‘Nanguo’ pears removed from cold storage and during the latter part of the experimental shelf period. The GABA shunt thus was prompted to assist in the normal TCA cycle operation in resistance to cold stress.

Mitochondria perform oxidative metabolism in eukaryotes and are the site of the ultimate oxidation of sugars, fats, and amino acids for release of energy. The mitochondrial matrix contains all the enzymes required for the TCA cycle, and the common pathway for final substrate oxidation in the mitochondria is the TCA cycle, followed by oxidative phosphorylation. Therefore, mitochondrial structure and membrane integrity play a vital role in maintaining normal plant metabolism to cope with environmental stress. In the current study, the integrity of mitochondria in untreated fruits was disrupted after 180 days of cold storage. GABA treatment has been shown to effectively maintain mitochondrial membrane permeability and structural integrity (Li Z. et al., 2019). From the perspective of GABA biosynthesis, the GABA synthesis-related key enzyme, GAD, was regulated by exogenous Ca²⁺ treatment. After Ca²⁺-treated fruit was placed in cold storage for 180 days, the mitochondrial structure in cells of the peel tissue was clear and complete. Consequently, the integrity of mitochondrial structure was maintained, and browning of ‘Nanguo’ pears was effectively alleviated.

Our results showed that exogenous Ca²⁺ delayed browning of the pericarp of ‘Nanguo’ pears upon removal from cold storage. The statistical analysis showed that exogenous CaCl₂ treatment increased Ca²⁺ and CML content in the fruit, while increasing GAD activity, resulting in an increase in GABA content. This increased the activity of GABA-T and SSADH, and accelerated GABA biosynthesis according to external stress. In addition, TEM revealed that the mitochondrial structure in the pericarp of fruits treated with CaCl₂ was clearer and more intact than that in the pericarp of the control fruits. Therefore, we concluded that CaCl₂ treatment improved the ability of pear fruit to resist cold stress by regulating the GABA shunt and reduced browning of the pericarp of ‘Nanguo’ pears after long-term cold storage ( Figure 6 ).

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

JL and SJ participated in the editing of the manuscript for this paper and the arrangement of the experimental process. BW and XZ provide help for the data analysis of the article. YZ and QZ supported the experimental operation. All authors of the study confirmed the accuracy of the content of the manuscript and the validity of the data.

This work was supported by the National Natural Science Foundation of China (31971698).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.