Ozone fumigation delays chlorophyll degradation in chili

Chlorophyll degradation plays a crucial role in mediating the senescence and devaluing of 'Jinda' chili fruit after harvest. The objective of this study was to investigate the effect of ozone fumigation on the chili fruit quality in relation to ROS-mediated chlorophyll degradation and cell death-related processes. Green chili fumigated with ozone (50 mg L?1) for 30 min was examined for antioxidant systems, chlorophyll degradation and cell death-related enzymes during 12 d of storage. The dissipation of pericarp degreening and reddening of pericarp color were observed on day 3 of storage, which were associated with the significantly elevated levels of superoxide and hydrogen peroxide accumulations, as well as increased chlorophyll catabolic enzymes (chlorophyll peroxidase, chlorophyllase and pheophytinase activities).? Moreover, both caspase-like enzymes and in situ DNA degradation, features of programmed cell death (PCD), became more obviously visible at the early period of deterioration. Fruit fumigated with ozone, enhanced the activity of antioxidant enzymes (ascorbate peroxidase, catalase and superoxide dismutase) and suppressed both chlorophyll degradation and the PCD process, which were compatible with lowering senescence in chili. Our findings showed that ozone fumigation increases antioxidant activity, which in turn helps to delay chlorophyll breakdown and cell death, hence slowing senescence in 'Jinda' chili. IntroductionChlorophyll breakdown is an integral part of plant senescence, a phenomenon accompanied by shorter life of fruit and green harvested crops (Yamauchi et al., 2004, Oda-Yamamizo et al., 2016). The plant chlorophyll catabolic process involves in various types of enzymes (Yamauchi et al., 2004, Dissanayake et al., 2012, Oda-Yamamizo et al., 2016). At the initial step, chlorophyll b (Chl b) is reduced to chlorophyll a (Chl a) and turned into chlorophyllide a (chlide a) by the sequential cascade of chlorophyll b reductase, hydroxymethyl catabolite reductase and chlorophyllase, respectively (Fayos et al., 2019). Subsequently, Pheophorbide a is generated through the removal of central Mg from chlide a catalyzed by Mg2+ dechelatase, resulting in a brown appearance. In the alternative ways, pheophorbide a formation is also propagated directly through transition of Chl a into pheophytin a as an intermediate catalyzed by Mg2+ dechelatase and pheophytinase enzymes (Aiamla-or et al., 2012). At the final step, pheophorbide a from those two pathways is turned into fluorescent chlorophyll catabolites by pheophorbide a oxygenase and red chlorophyll catabolite reductase, respectively (Aiamla-or et al., 2012). Fluorescent chlorophyll catabolites are transformed to nonfluorescent chlorophyll catabolite or nonfluorescent dioxobilin-type chlorophyll catabolite, which is final-products of chlorophyll degradation (Oda-Yamamizo et al., 2016, Fayos et al., 2019). Besides the pheophorbide a oxygenase pathway, peroxidase (POX)-mediated chlorophyll degradation or chlorophyll peroxidase also plays a vital enzyme in catalyzing the dissipation of chlorophyll. During peroxidase-mediated chlorophyll degradation, the POX reacts with H2O2 to generate phenoxy radicals from phenolic compounds. These radicals then react with chlorophyll and its derivatives, leading to the formation of various colorless, low molecular weight compounds via the production of intermediates such as the C13?-hydroxychlorophyll a, fluorescent chlorophyll catabolite and some bilirubin-like compounds (Yamauchi et al., 2004). The ascending activity of the chlorophyll catabolic enzymes was found to accelerate senescence in many harvested crops. The appearance of dark spots on banana peel was associated with chlorophyll degradation, as indicated by elevated levels of chlorophyllase, pheophytinase, Mg-dechelatase and chlorophyll degradation peroxidase (Pongprasert et al., 2021). The expression level of the chlorophyll degradation-related genes, chlorophyllase and pheophorbide a oxygenase, was associated with the loss of fruit firmness and discoloration in yellow-pear cultivar (Charoenchongsuk et al., 2015). However, the treatment in retarding chlorophyll degradation such as gibberellic acid (Xiao et al., 2022), mannose (Guo et al., 2022) and heat treatment (55 ?C) (Kaewsuksaeng et al., 2015) via the suppression of chlorophyll catalytic enzymes has been shown to delay plant senescence and physiological disorders. Reactive oxygen species (ROS) are the active oxygen molecules including O2?-, hydrogen peroxide (H2O2) and hydroxyl radicals generating as the byproduct of oxygen consumption from cellular respiration in mitochondria and leakage of electron during photosynthesis in chloroplast (Sharma et al., 2012). Excessive ROS buildup results in oxidative damage of the cellular biomolecules such as lipid, protein and nucleic acid, eventually leading to cellular malfunction, organelle disorganization and cell death. Many senescence symptoms such as banana peel spotting (Chotikakham et al., 2022a), litchi pericarp browning (Siddiqui et al., 2021) and longan pulp break down (Lin et al., 2021), have been linked to ROS accumulation and oxidative membrane damage (as indicated by malondialdehyde, MDA). Interestingly, the production of harmful ROS resulting in oxidative damage was found to accelerate the detrimental effects on chlorophyll and chloroplast structures (Yamauchi et al., 2004; Hu et al., 2020). ROS was found to be attributed to promote yellowing in broccoli (Nomura et al., 2017) and pak choi (Brassica rapa) (Song et al., 2020). To limit ROS formation, restore oxidative damage and to get rid of other drastic effects, plants have evolved effective antioxidant enzymes such as ascorbate peroxidase (APX), catalase (CAT) and superoxide dismutase (SOD) (Sharma et al., 2012). Previous studies have demonstrated that treatments such as 1-methylcyclopropene, putrescine and glycine betaine significantly reduced or attenuated senescence symptoms such as flower wilting in sword fern (Nephrolepis cordifolia), leaf yellowing in pak choi and chilling injury in hawthorn fruit during storage via enhancing antioxidant enzymes and delaying ROS production as well as oxidative damage (Razavi et al., 2018, Song et al., 2020, Qu et al., 2020). Programmed cell death (PCD) is not only the final stage in plant development, but it is also required for the successful elimination of unwanted or senescent tissue, so controlling PCD activation in proper level has become a key mechanism in delaying the deterioration (Van Doorn, 2004, McIlwain et al., 2013). In plant system, PCD is processed by various kind of cysteine-aspartic proteases or caspase-like enzymes. Upon the caspases activation, the cell death characteristics are created. Among the caspase enzymes, caspase 3 plays a significant role in the committed step of PCD process. An active caspase 3 promotes specific endonuclease enzyme (caspase activated DNase) causing DNA degradation and allowing 3?-hydroxyl terminus exposure to be detected using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) as a cell death hall mark (McIlwain et al., 2013). Caspase 3 also causes the instability of the plasma membrane by disrupting flippase function, causing phosphatidyl serine externalization (Nagata et al., 2016). Moreover, some situations indicated that if the PCD does not proceed, the senescence can be retrospective, as indicated by yellowing leaves that can regreen again under cytokinin exposure (Rapp et al., 2015). Treatments effectively retard PCD have been shown to delay senescence in many harvested crops. Sucrose alleviated PCD in broccoli after harvest by lowering caspase 1, 2, 3, 4, 6 and 8-like activity as well as delaying TUNEL positive nuclei (Li et al., 2020). The lowering in caspase 3-like activity was compatible with the reduction in peel spotting of banana under methyl salicylate immersion (Chotikakham et al., 2022b). Glycine betaine treatment reduced caspase 3 activity and maintained membrane fatty acid metabolism in cold-stored peaches (Wang et al., 2019). Chili (Capsicum annuum L.), the most extensively eaten spice, is utilized as seasoning in cooking in countries over the world. In Thailand, ?Jinda? chili is a hot chili pepper cultivar that is frequently used in a number of Thai recipes (Ahmed et al., 2000, Ran et al., 2019). Unfortunately, chili is a perishable agricultural crop due to the rapid dissipation in the greenness of pericarp peel color after harvest which reduces the nutrition, natural active compounds and marketable value (Ahmed et al., 2000, Fayos et al., 2019, Ran et al., 2019). Thus, the approaches to extend chlorophyll retention are critical in prolonging the fruit?s economic value. Our recent research has shown ozone fumigation to be an effective method in improving fruit quality and storage life (Janhom and Whangchai, 2022). However, the physiological mechanism of ozone in extended fruit storage life still not known. Therefore, this study aimed to explore the underlying strategies of ozone fumigation in relation to ROS-mediated chlorophyll degradation and cell death of ?Jinda? chili during storage. SourcesOzone fumigation promotes antioxidant activities to retard chlorophyll degradation and cell death in ?Jinda? chili during storageNatthapong Janhom & Kanda WhangchaiPostharvest Biology and Technology,?Volume 202, August 2023, 112375https://doi.org/10.1016/j.postharvbio.2023.112375https://www.sciencedirect.com/science/article/abs/pii/S0925521423001369 Picture by Cat's Thai Kitchen, https://www.facebook.com/107200047451964/posts/134435988061703/