Effect of exogenous ascorbic acid on blackheart occurrence and anti-oxidation activity in postharvest pineapple

Abstract:【Objective】Pineapple is an important tropical fruit in China, whose planting efficiency and export trade can directly affect the economic income of farmers in tropical regions. However, the pineapple of postharvest is prone to blackheart during the process of storage and transportation. The cell membrane structure of diseased fruit is destroyed because of excessive ROS accumulation, in which the spatial distribution of peroxidase and phenolic substances are disrupted. The combination of enzymes and substrates and the oxidation of phenols to quinones lead to the fruits browning.'Paris' pineapples (Ananas comosus'Comtede Paris') are taken as the test materials in the study to investigate the effect of exogenous ascorbic acid on blackheart occurrence and anti-oxidation activity of postharvest pineapple.【Methods】The bottom of the'Paris' pineapples fruits were soaked in 0.5 mg· L-1 Miramide for 1 min and dried in the air. Then, 'Paris' pineapples fruits were divided into A and B groups, 15 fruits in each group. Group A and group B were put into clear water and 0.2% (mass volume fraction) ascorbic acid (L-ascorbic acid, As A) for 15 minutes respectively, clear water as control. After air drying, they were put into the 0.02 mm thick polyethylene perforated film bag and stored in the fruit storehouse under the condition of (25±2) ℃ and relative humidity 85%-95%. By observing and recording the pheno-typic changes of blackheart occurrence on the 3, 6, 9, and 12 days after treatment, the effect of exogenous ascorbic acid on the occurrence of blackheart disease were analyzed. In order to determine the antioxidation capacity of postharvest pineapple after the treatment of exogenous ascorbic acid, the fleshes close to the fruits core by different treating time were taken to measure the key enzyme activity by UV2700 instrument and analyze its correlative changes. Based on previous transcriptome sequencing results in our lab, the local blast library was built. APX gene sequences (LOC109724021 and LOC109708835) and GR gene sequences (LOC109715444) registered on NCBI were used as query sequences and then compared under the condition of e<1e -5 and coverage > 60%. Finally, 16 APX genes and 5 GR genes were obtained. Under the homogenized condition of room temperature storage, their expression profiles were obtained. On the basis of previous research works, the study analyzed the transcriptional expression level of key enzyme genes in the enzymatic/non-enzymatic antioxidant systems using Me.V 9.0 and LightCycler® 480 q-PCR instrument.【Results】Exogenous ascorbic acid could effectively delay the occurrence of blackheart and reduced the disease index of pineapple blackheart. In the control groups treated with clear water, the blackheart disease was observed on the third day, in which disease index was 13.34%. However, in the experimental groups treated with exogenous ascorbic acid, the blackheart disease appeared on the 6th day. After treatment for 12 days, the fruits core and most of the flesh in control groups completely became dark brown in the control groups, but in the experimental groups, browning only appeared on the flesh area near the fruits core, in which the disease index had significant differences and reached 95.56% and 53.33%, respectively. During the entire storage periods, ascorbic acid treatment could significantly reduce the H2O2 content of pineapple fruits, especially in the later stages of processing. H2O2 content in experimental groups was decreased by 20.74% than that in the control groups after treatment for 12 days. Ascorbic acid treatment also significantly inhibited the decrease of AsA content, increased the content of reduced glutathione (GSH) in pineapple fruit, maintained the high content level of AsA and GSH for at least 9 days. After 9 days, the AsA and GSH content were both reduced to the same level as the control groups. The results of enzymic activity analyses showed that ascorbic acid treatment could significantly improve the activity of ascorbate peroxidase (APX) and glutathione reductase (GR). After treatment for 12 days, the activities of APX and GR in experimental groups were respectively increased by 48.51% and 12.49% than that in control groups. In addition, the activity of CAT enzyme in the experimental groups and in the control groups all showed a continuously increasing trend, but the activity value in control groups was bigger than that in the experimental groups at each detection time points. The physiological and biochemical mechanisms that caused this result needed further exploration. The result of heatmap analysis and the real-time fluorescence quantitative PCR showed that APX and GR genes were involved in the response to blackheart diseases, and were induced or inhibited in different degrees. The expression patterns of APX5, APX12, and GR5 genes, whose transcriptome expression profiles were consistent with real-time fluorescence quantitative expression results at room temperature, were analyzed under ascorbic acid treatment by real-time fluorescence quantitative PCR. The results showed that they all respond to ascorbic acid treatment in different degrees.【Conclusion】The treatment of exogenous ascorbic acid could effectively delay the occurrence of blackheart diseases, enhance the capacity of ROS scavenging and the key enzyme activity of antioxidant defense system. In the progress of blackheart occurrence, the key enzyme genes responded to the treatment of ascorbic acid in different degrees. The results indicated exogenous ascorbic acid could enhance antioxidative activities of pineapple fruits and delay the blackheart occurrence of postharvest pineapple fruits and extend the storage time of postharvest pineapple fruits.