Cold storage is a widely adopted post-harvest technique, yet it poses challenges for sensitive commodities due to undesirable effects on texture, taste, peel color, and cellular activities triggered by physiological, physical, and biochemical processes (Aslam et al. 2023). Papaya belongs to the cold sensitive fruit, one of the main challenges in preserving papaya fruit is preventing chilling injury (CI), which can cause surface pitting, scalds, and premature ripening when the fruit is stored at temperatures below 10 ℃ (Pan et al. 2017). To address this, we employed cold shock treatment, demonstrating its positive impact on increasing antioxidant enzyme activity and alleviating CI symptoms.
In our study, we investigated the impact of cold shock treatment (CST) on physiological and morphological changes associated with cellular injury (CI). Initially, chilling injury spots were noted in the stalk area of the control fruit, indicating susceptibility to cold stress. In contrast, the fruit subjected to CST did not display any signs of injury. As the storage period progressed, the number of chilling injury spots on the control fruit steadily increased, while the CST fruit remained unaffected by such damage (Fig. 1). Our findings support previous studies showing that CST treatment effectively alters the physiological responses to chilling stress and reduces the occurrence of CI in various species like gingers (Zhang et al. 2023), cucumbers (Zhao et al. 2018), and bananas (Wu et al. 2014). The occurrence of CI is primarily attributed to changes in the structure and conformation of cell membranes, which significantly impact their permeability. When cells are exposed to low temperatures, their cell membranes can become damaged, resulting in an increase in the release of intracellular electrolytes (Liang et al. 2020, Huang and Wang 2023).
The cold storage period had an impact on the cell structure of both control and CST papayas.
Our results reveal alterations in the cell structure of both control and CST papayas during the cold storage period. Control fruit exhibited faster ripening with cells fusing together, while CST fruit showed a slower ripening process with some signs of loosening and irregularity in cell structure, maintaining relatively intact cell outlines (Fig. 1A). Therefore, CST papaya exhibited a delay in cellular breakdown compared to control, indicating its potential for longer storage periods.These findings align with previous research (Lin et al. 2017; Sun et al. 2020).
In this study, we found that chilling injury index (CII) increased gradually over time in both control and CST papayas at low temperature, with a more pronounced increase in control papayas. CST papayas displayed lower relative electrolyte leakage (Fig. 1C), indicating a protective effect against membrane permeability and a positive impact on maintaining structural integrity during storage. The decreased cell membrane permeability positively correlated with cold resistance and reduction of chilling injury in various fruits like guavas (Lo’Ay and Taher 2018), nectarines (Ali et al. 2023) and peaches (Yang et al. 2014; Zhang et al. 2020; Wang et al. 2021), suggesting the potential of CST to delay CI development in papaya and extend its shelf life.
The biological function of COR gene has been extensively studied for its ability to enhance the plant's resilience and adaptability to cold stress. Our study delves into the molecular mechanisms by examining the impact of CST on COR gene expression in papaya fruit during storage. Transcriptome and qPCR analyses revealed significant changes in the transcript levels of COR genes, with CST upregulating the expressions of CpCOR1, CpCOR2, and CpCOR3 genes (Fig. 2). This is consistent with findings in wheat and Arabidopsis (Yokota et al. 2015; Zhou et al. 2018), emphasizing the positive role of COR genes in enhancing cold tolerance under low-temperature stress.
In the present study, CpCOR1 has been proved to be a member of COR PM gene family, and it is closely related to homologous genes from other species (Fig. 3). The presence of GFP tag in the inner epidermal cells of onion provided evidence that CpCOR1-GFP, a protein that responds to low temperatures, and confined on the plasma membrane. Similarly, Breton et al. (2003) demonstrated that COR413-PM plays a crucial role in ensuring the stability of the lipid bilayers within the plasma membrane. Thalhammer and Hincha (2014) suggested that COR15PM proteins play a significant role in stabilizing the plasma membrane in extreme environments of high salt and cold temperatures. The functions of COR413 proteins in Arabidopsis thaliana, including COR413IM, COR413PM, and COR413TM localized in different membranes, have been studied under cold stress (Shi et al. 2018). These findings suggest that CpCOR1 is a transmembrane protein to play crucial role in maintaining membrane integrity during stress through modifying the structure of membrane lipids.
To deepen our understanding of CpCOR1's biological function, we conducted transient overexpression experiments. Both heterologous and homologous results demonstrated that CpCOR1 overexpression effectively reduced yellowing, morphological changes, and enhanced fruit quality. This was accompanied by a decrease in membrane permeability and malondialdehyde (MDA) levels and an increase in antioxidant enzyme activities such as catalase (CAT) and ascorbate peroxidase (APX) (Figs. 5, 6). Previous studies have demonstrated that the activation of expression of COR genes can significantly enhance the resistance to various stress, for example, SikCOR413PM1 gene in transgenic tobacco plants can enhance their resilience to extreme conditions such as low temperatures and water scarcity (Guo et al. 2019). Zhang et al. (2021) revealed that overexpression of LeCOR413PM2 resulted in enhanced cold tolerance in tomato plants through protection against cell membrane damage, reduction of ROS accumulation. Conversely, suppression of LeCOR413PM2 expression through RNA interference could led to increased cold sensitivity in plants (Zhang et al. 2021). Moreover, the positive effects of COR genes on avoiding the harsh environments closely associated with the enhancement of antioxidant enzyme activities, including CAT, SOD, APX, POD, GR, and others (Huan et al. 2017). Our results once again prove that the enhancement of antioxidant capacity could contribute to improve cell membrane stability and stress tolerance in plant.
In plant, the ICE1-CBF/DREB1-COR pathway is a well-known and predominant signaling pathway that plays a crucial role in regulating cold stress responses (Shin et al. 2017). Although the COR gene plays a direct role in this pathway, increasing evidence has indicated that the other pathways and genes involve in the cold response in fruits and vegetables. For example, Liu et al. (2020) revealed that the GSH-associated genes (GST Tau, MAAI, APX, GR, GS and MDHAR) were significantly up-regulated in NaHS-treated cucumber seedlings, compared to the H2O-treated seedlings under chilling stress. In tomato fruit, overexpression of Sly-miR167a delayed postharvest chilling injury under low temperature storage (Li et al. 2023). Yu et al. (2023) suggested ethylene signaling component, SlERF2 contributed to the regulations of ABA biosynthesis and signaling, as well as CBF cold signaling pathway, ultimately affecting the fruit quality during long-term cold storage. Some transcript factors, such as MaMYB13 (Li et al. 2023), and VaNAC72 (Qin et al. 2023) are involved in response to chilling stress and positively regulate cold tolerance in banana and grape (V. vinifera). However, Cao et al. (2023) found that PpRAP2.12 functions as a negative regulator of plant defenses against chilling stress, and may provide a target for mitigating chilling injury in peach fruit. Thus, further exploration is needed to understand the interactions between COR genes, transcription factors, and other pathways, enhancing our knowledge of cold response regulation and fruit quality during long-term cold storage.
Figure 7 depicts the proposed model of cold shock treatment and CpCOR1 regulating cold tolerance and antioxidant activities in papaya fruit. Cold shock treatment significantly reduces chilling injury in papaya by maintaining cell structure integrity, decreasing membrane permeability, and reducing MDA accumulation during storage at 5 ℃. CpCOR1, identified as a basic hydrophobic protein belonging to the COR413 subgroup, plays a crucial role in alleviating chilling injury and delaying ripening by enhancing antioxidant activities in papaya fruit under postharvest chilling stress. The use of cold shock treatment emerges as a promising technique for increasing chilling resistance in harvested fruit. These findings lay the groundwork for further investigations into the upstream regulatory factors of CpCOR1 and the network of cold response during storage of fruit.