Effects of methyl salicylate on programmed cell death via reactive oxygen species-triggered mitochondrial pathway involved in reducing senescent spotting of ‘sucrier’ banana fruit during storage

Abstract

Mitochondria are the major source of cellular reactive oxygen species (ROS) production. These highly reactive molecules play an important role in instigating senescence and physiological disorder in plant. An imbalance between mitochondrial ROS production and scavenging can disrupt mitochondrial physiological properties and functions, leading to programmed cell death (PCD) activation through leaked Cytochrome c (Cyt c). Senescent spotting is the major postharvest physiological disorder that spontaneously occurs during the ripening of ‘Sucrier’ banana (Musa acuminate). The symptom shortens in both storage life and reduced market value. Until now, the mechanism responsible for the development of peel spotting is still not known. In addition, the detailed involvement of ROS on mitochondria-mediated PCD in mediating peel spotting is not totally understood. This study aimed to demonstrate the relationship between ROS and mitochondria-mediated PCD in relation to peel spotting and to evaluate the mechanism of methyl salicylate (MeSA) in the reduction of peel spotting via controlling the mitochondria-mediated PCD pathway. The effect of MeSA treatment of ‘Sucrier’ banana on ROS and mitochondrial physiological properties and functions in relation to senescent spotting development during storage was first examined. The ripen banana at color index 4 was immersed in either 0.25% ethanol (control) or 2 mM MeSA (experiment) for 30 min and stored at 251 °C with 75% relative humidity for 6 d. MeSA effectively hindered the occurrence of senescence spotting during the storage. At the same time, both mitochondrial superoxide, hydrogen peroxide and calcium ion (Ca2+) accumulation were found to be reduced. This reduction of ROS and Ca2+ levels coincided with enhanced activity and expression of mitochondrial antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, ascorbate peroxidase and alternative oxidase. The treatment lowered overall permeability of mitochondria, as demonstrated by delaying the opening of mitochondrial permeability transition pores (MPTP) and preventing the loss of mitochondrial membrane potential and mitochondrial Cyt c. Moreover, mitochondrial functions as indicated by the activity of NADH dehydrogenase, succinate dehydrogenase and cytochrome c oxidase activities as well as ATP content remained higher under the treatment. These findings suggested that MeSA treatment maintained mitochondrial physiological properties and functions through strengthening mitochondrial antioxidant defense mechanisms which ultimately leads to postponing the onset of peel spotting. The effect of MeSA on mitochondria-mediated programmed cell death in relation to senescent spotting development of ‘Sucrier’ banana during storage was then examined. The ripen banana at color index 4 was treated as mentioned above. The results established a clear connection between PCD and peel spotting development. During the development of peel spotting, prominent PCD markers, such as caspase-like activity, in situ DNA fragmentation and cell death were found concomitantly with the development of spotting lesion. MeSA treatment reduced mitochondrial Cyt c leakage and activated the expression of anti-apoptotic gene (14-3-3ι). The decreased Cyt c leakage and enhanced expression of this gene gave rise to lowering of caspase 8, 9 and 3-like activities and DNA degradation. Late PCD markers such as DNase activity and poly (ADP-ribose) polymerase cleavage, proteolytic activity (protease activity, protein and amino acid contents) and propidium iodide uptake were found to be minimized as well. The findings suggested that MeSA retards mitochondrial Cyt c leakage and upregulates the expression of anti-apoptotic genes, thus, preventing mitochondria-mediated PCD activation, thereby mitigating the peel spotting of ‘Sucrier’ banana. These results indicated that the occurrence of peel spotting is associated with ROS-triggered mitochondria-mediated PCD process. MeSA treatment enhanced antioxidant defense systems and anti-apoptotic gene expression, leading to improved mitochondrial physiological properties and functions, subsequently postponing PCD, thereby reducing peel spotting of ‘Sucrier’ banana during storage.