Analysis Of APX And Cat Enzymes’ Activities Of The Stored Fruits

Cold temperature is one of the different stress conditions that can interfere with the equilibrium in the production of oxidants and its scavenging ability by antioxidant enzymes. Thus the survivability or responses of plants under cold temperature can be influenced by duration of the storage. We have studied the performances of Punica granatum (cv ‘Wonderful’) stored under temperature of 6. 5 °C for a period of one month by evaluating the amount of activated reactive oxygen species (ROS). Reactive oxygen species including H2O2 are comparatively stable. Even though, H2O2 was reported to be beneficial in lower amount, its uncontrolled aggregation may promote the production of a more toxic compound called hydroxyl radical (OH-). Hydroxyl radical is a highly reactive and the most sensitive ROS known with its ability to cause oxidative degradation of lipids, disruption of membrane or cellular death. In mitigating the ROS induced destruction stated above; plants defend themselves from via the activation of antioxidant enzymes. Among these enzymes is the APX which function in the degradation of H2O2 to form water and oxygen by utilizing the oxidized ascorbate as an electron acceptor. CAT being another enzyme though having a similar function to APX is known for its role in the degradation of H2O2 (stress indicator) to oxygen and water. Thus in this study, the activities of APX and CAT enzymes were also investigated to measure the free radical scavenging capacity of the stored fruits.

Being the main cause of membrane disruption in plant tissue, the extent of lipid peroxidation in the fruit was measured by evaluating the MDA content of the stored fruit. In the present study, storage temperature considerably had an effect on the production MDA content. The MDA content was notably higher in the seed obtained from fruits stored at 6. 5 °C temperature compared to that obtained from the fresh fruits. This implies that cold tolerability in plants may be reflected by the level of MDA. Furthermore, lipid peroxidation may be one of the first evidence of chilling injury, because increased MDA aggregation disrupts the cell membrane. The result for MDA content in the present study corroborates with the previous findings by Aghdam et al. (2012) where an increase in MDA content of tomato fruit treated with 0. 002 M of salicylic acid was reported after a-three week period of storage at 1 °C. A previous study by Imahori et al. (2008) on chilling-induced oxidative stress had earlier reported a sharp increase in the MDA level of Mume fruits stored under a temperature of 6 °C within five days before it decreased gradually. Similarly, Hordijk (2013) found that the longevity of cold storage in citrus fruit could cause an increment in MDA level as a result of a rise in the oxidative membrane damage with occurrence of chilling injury. Furthermore, Kumar (2017) recently observed a more than 3-fold increment in the kinetic of MDA production in plum fruits that were dipped in distilled water (control) when compared with those that were surface-coated with lac prior to cold storage for about 35 days. Thus, concluding that surface-coating of fruits prior to cold storage may be efficient in reducing its MDA level with storage temperature.

In contrast to these findings, a significant increase in MDA contents of strawberry fruits under cold storage was reported and this was attributed to the observed accumulated reactive oxygen species in the fruit under the same conditions. Hydrogen peroxide (H2O2) is a toxic substance with the ability to induce membrane disruption and thus resulting into lipid peroxidation. Moreover, H2O2 can function as an indicator controlling many activities in plants. In this present study, a slight but not significant increase in H2O2 level was observed after storage of pomegranate fruits for a period of one month at 6. 5 °C and compared with the level of the control. This shows that the controlled and stored fruits had the same phenomenon for the accumulation of reactive oxygen species. This result was in agreement with the findings of Rosalie et al. (2018) where values with no statistical difference were reported in the H2O2 level of ripe mango fruits (cv. ‘Cogshall’) stored differently at 7, 12 and 20 °C for about 2 weeks. A little difference was also reported in the level of H2O2 during storage of peach fruits at 5 °C for ten days. However, these findings were not in agreement with Dokhanieh et al. , (2016) where a significant decrease in was reported in pomegranate arils treated with salicylic acid prior to storage at 4 °C for a period of two weeks when compared to the control Dokhanieh et al. , (2016).

In another study, Peng et al. (2017) reported a considerable increase in the accumulation of H2O2 in strawberry fruits throughout its cold storage temperature. Under stress conditions, the synergetic activities of SOD, APX and CAT which neutralises the harmful O- radicals and H2O2 can be effective in the elimination of the reactive oxygen species. These ultimately defend the plants against a-more cellular damage from toxic radicals. Ascorbate peroxidase (APX) employs ascorbate as an electron donor in the reduction of H2O2 to H2O, thus preventing the aggregation of H2O2 to a harmful level during stress (Uthairatanakij et al. , 2016). In this present study, two distinct APX isozymes were observed on the native page. APX 1 shows a slight but not significant increase in the activities of the fresh fruit when compared to the observed band corresponding to the stored fruits. In APX 2, a more intense band was observed with a statistical difference in the stored fruit when compared to the control.

The observed intensity of APX 1 shows that both conditions have relatively the same disproportionation ability. However, up regulation of the isozyme in APX 2 can be attributed to a higher tendency of the stored produce to dismutate the accumulated hydrogen peroxide in the stored pomegranate fruit. Since the stored fruits showed a higher H2O2 value and MDA content, indicating oxidative disruption; the up regulation of APX 2 observed in the stored fruits can therefore be linked with the fruits’ response to neutralize the accumulated ROS.

Moreover, it can be explained that in the stored pomegranate fruits, APX isoforms were produced to eliminate the excess H2O2 and thus protecting the fruits from ROS. In addition total APX activity proved a higher (but not statistically different) total APX activity in the fresh fruits when compared with the treated. A no-significant difference among the APX activities of pineapple fruits (dipped into salicylic acid prior to storage at 10 °C for 20 days) and the control was reported by Lu et al. (2010). In contrast, Dokhanieh et al. (2016) reported a considerable increase in APX activity of pomegranate arils in fruits dipped into an hot salicylic acid prior to storage at 4 °C for about 2 weeks when compared with the control.