Department of Horticulture, College of Agriculture, G. Different antioxidants and salicylic acid were tested to overcome pericarp browning and to maintain the postharvest quality of the litchi fruits at ambient storage. It was found that 0. Application of 0. It was effective in reduction of polyphenol oxidase activity and improvement of anthocyanin pigments of the fruit pericarp over other treatments. Therefore, 0.
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Polyphenol oxidase PPO plays a key role in the postharvest pericarp browning of litchi fruit, but its underlying mechanism remains unclear. In the pericarp of newly-harvested litchi fruits, the LcPPO expression level was relatively high compared with developing fruits.
Regardless of the litchi cultivar and treatment conditions, the LcPPO expression level and the PPO activity in pericarp of postharvest fruits exhibited the similar variations. When the fruits were stored at room temperature without packaging, all the pericarp browning index, PPO activity and the LcPPO expression level of litchi pericarps were reaching the highest in Nandaowuhe the most rapid browning cultivar , but the lowest in Ziniangxi the slowest browning cultivar within 2 d postharvest.
Preserving the fruits of Feizixiao in 0. Thus, we concluded that the up-regulation of LcPPO expression in pericarp at early stage of postharvest storage likely enhanced the PPO activity and further accelerated the postharvest pericarp browning of litchi fruit.
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Competing interests: The authors have declared that no competing interests exist. Polyphenol oxidases PPOs are a group of metalloproteinases encoded by nuclear genes and capable of binding copper. In general, catechol oxidase and laccase are collectively referred to as PPOs. PPOs can catalyze oxidation of the monophenolic hydroxyl group and dihydric phenol to o -diphenol and o -quinone, respectively.
Quinones can self-polymerize in the plant body or react with amino acids and proteins in the cell, leading to the formation of black or brown substances and the browning of plant tissue. This reaction is considered to be the main reason for the postharvest browning of fruits and vegetables. Litchi Litchi Chinensis Sonn.
In the year of , the planting area of litchi was approximately , hectares and the fruit yield was 1. However, the rapid postharvest browning of pericarp reduced the commodity value of the fruit and limited the expansion of the litchi trade, and was considered as the most important limitation to the continued development of the litchi industry  — . Li and his colleges first proposed that litchi pericarp browning is an enzymatic browning process caused by PPO activity . Furthermore, several reports demonstrated that the partially purified PPO enzyme could only oxidize polyphenols containing the ortho - hydroxyl group, suggests that the enzyme lacked cresolase and laccase activities and was a diphenol oxidase  — .
When a reaction was carried out with the natural substrate, the phenols isolated from litchi pericarp, and partially-purified litchi pericarp PPO, the brown substance s formed  , . Further research indicated the pericarp contains a variety of phenols; most of them could be oxidized by PPO .
The - -epicatechin  and procyanidin A2  occurring at relatively high levels in pericarp are the direct substrates of PPO. Additionally, PPO oxidizes anthocyanin in the presence of polyphenol, resulting in the formation of brown substances .
To take together, these findings confirm that pericarp contains the substrates of PPO. The distribution range of PPO activity in pericarp is generally consistent with that of phenols. Both of them are present in the epidermis and sclerenchyma cells of exocarp and some of the cells of the mesocarp and endocarp  , .
The PPO activity in the easily-browned exocarp is higher than the slightly-browned mesocarp and endocarp . During the postharvest aging process, the pericarp PPO activity diffuses in proportion with the browning site, consistent with the browning process . When the PPO activity is inhibited by fruit treatments such as cold storage, chemical preservative, and coating, the pericarp browning process is also slowed down  , .
In contrast, when the PPO activity is enhanced by fungal infection  or ethylene treatment  , the pericarp browning is accelerated. Therefore, phenol oxidation catalyzed by PPO in pericarp may be a direct cause of pericarp browning, and reduction of the PPO activity in pericarp is expected to inhibit pericarp browning.
For example, the PPO activity and browning potential are reduced in apple caulicles and calluses, and this process is associated with the expression of PPO antisense RNA . Expression of potato PPO antisense RNA in tomato reduces the levels of tomato PPO gene expression and PPO activity, further reduces the capacity for oxidation of the caffeic acid in tomato leaves by approximately folds .
Initially, we hypothesize that specific inhibition of PPO expression in litchi pericarp would suppress postharvest pericarp browning. Our paper proved solid data which support that LcPPO expression is up-regulated and the PPO activity is enhanced at an early stage of postharvest storage, possibly leading to the acceleration of pericarp browning.
Our conclusion can facilitate the further elucidation of the mechanism of litchi pericarp browning which can be applied to the breeding of long-storage litchi cultivars by using bio-engineering techniques.
In the year of , litchi fruits of three different cultivars, Feizhixiao, Ziniangxi, and Nandaowuhe were obtained from a test base of the Chinese Academy of Tropical Agriculture Sciences. Fruits with the same maturity of Ziniangxi and Nandaowuhe were harvested on June 7 th of All fruits were transported to the laboratory within 3 h after the harvest and sorted immediately by selecting those of equivalent maturity, without pests, diseases, and mechanical injuries.
The fruits 20 fruits per group were processed in triplicate groups. Semi-lignified spring shoots and their young leaves, unopened flower buds, and the absorbing roots of seedlings of Feizixiao were collected respectively. The pericarp was peeled from fruits of Feizixiao, Ziniangxi, and Nandaowuhe cultivars after different treatments.
The level of pericarp browning and the PPO activity as well as the LcPPO expression in different litchi cultivars was evaluated in the laboratory. Afterwards, the containers were covered with lids while maintaining a 1-cm gap. At the beginning, the fruits of Feizixiao were placed in 0. Specimens were taken out after the day of the harvest day 0 and days 1, 2, 3, 5, and 7 postharvest.
The fruits of Feizixiao from the previous experiment were used as control. The fruits of Feizixiao were placed in preserving containers in a single layer without squeezing and the containers were closed as described above. The fruits of Feizixiao from the first experiment were used as control.
The pericarp must be carefully peeled from all fruit samples to avoid adhesions of pulp or juice. Two grams of pericarp samples was weighed and placed into a clean mortar, about 8 mL of phosphate buffer 0. After centrifugation, the supernatant was applied as the crude enzyme extract. To determine PPO activity, total 3. Catechol was mixed with PBS in advance, and then the crude enzyme extract was added in.
One unit of enzyme activity was defined as the increase of A by 0. The protein concentrations were determined by the dye-binding method . Genome-walking amplification was performed using a commercial kit following the instructions of manufacturer TaKaRa, Dalian. The integrity and length of the PCR amplification products were estimated by running 1.
Eventually, the positive clones were also chosen and subsequently subjected to DNA sequencing. The similarity of the determined amino acid sequence with the PPO amino acid sequences of other biological species was compared using MEGA 5 method . In the southern blot assay, probe preparation, hybridization, and membrane washing were performed with a Roche DIG-labeling kit Roche, Shanghai according to the manufacturer's instructions.
After amplification, the data from the reaction plate were added to the research plate for processing. The LcPPO gene expression level in pericarp of newly-harvested Feizixiao fruit was used as the reference. All experiments were performed three times, and the arithmetic mean of replicate data were calculated and used for the comparisons. After experiment, all data were analyzed in the DPS data processing system . The resulted data were presented in the text.
The fragment was purified and sequenced; new primers were designed based on the sequencing data and used for genome-walking. The resulted target sequences have bps and bps in length respectively. The ORF is bps in length and encodes a polypeptide with amino acid residues.
The deduced polypeptide has M r 67, The instability parameter was To our knowledge, this is the first report of the LcPPO sequence. The polygenetic tree was drawn based on the NJ methods, which were described in Mega 5. Numbers at the branches represent percentage bootstrap support with replicates. The genomic DNA of Feizixiao was analyzed by southern blotting. By using the LcPPO -specific probes, only one clear band remained visible.
A clear band was visible in lane 1 and lane 2. The LcPPO expression level was the highest in the flower and the leaf, followed by the seed and the root and the young stem, pericarp and pulp the lowest; Fig. The highest expression was found in the flower and leaf, and the lowest expression was in the pulp. The LcPPO expression level of litchi pericarp was generally low and stable during fruit development, but increased rapidly after harvest.
During postharvest storage, the increasing of LcPPO expression level in the pericarp was consistent in various cultivars, i. It is noted that this is different from previous findings in apricot and banana during fruit development, which showed that PPO gene expression ceased in the color-turning stage in apricots  and decreases in peel of banana in the ripening stage .
Thus, the PPO gene expression in fruit tissues is species-specific. Fruits of Feizixiao were harvested on May 14 th , ; Ziniangxi and Nandaowuhe were harvested on July 7 th , Expression of LcPPO in pericarp increased sharply after harvested. Similar changes were observed with Feizixiao, Ziniangxi and Nandaowuhe. During postharvest storage, the pericarp-browning index of different litchi cultivars gradually increased over time.
Overall, Nandaowuhe showed the fastest pericarp browning and turned completely browned on day 4 after harvest, whereas Feizixiao turned completely browned in day 5 after harvest.
Ziniangxi showed the slowest pericarp browning and the color turned completely browned on day 7 after harvest Fig. The browning index values of the three litchi cultivars rapidly increased during 1—4 days after harvest. Within the same storage period, the pericarp-browning index values of Feizixiao and Nandaowuhe were constantly greater than Ziniangxi, while the values of Nandaowuhe were slightly larger than Feizixiao Fig. Pericarp-browning index A of Nandaowuhe increased fast compared with Feizixiao and Ziniangxi.
During postharvest storage, the pericarp PPO activity of the three cultivars increased at the beginning, but decreased afterwards.
The highest rate of increase was observed in Nandaowuhe, followed by Feizixiao, and Ziniangxi. The pericarp PPO activity reached the peaks in Feizixiao and Nandaowuhe on day 2 after harvest, however the enzyme activity was lower in the former cultivar than in the latter. In Ziniangxi, it appeared as a peak on day 3 after harvest. Within the same storage period, Nandaowuhe had the highest pericarp PPO activity on day 2 after harvest, followed by Feizixiao and Ziniangxi Fig.
The differences in postharvest pericarp browning of various litchi cultivars are related to the pericarp PPO activity at an early stage of storage. These findings demonstrate that the differences in the pericarp browning process among various litchi cultivars are positively correlated with the LcPPO expression level at the early stage of postharvest storage.
Control of post-harvest pericarp browning of litchi (Litchi chinensis Sonn).
Polyphenol oxidase PPO plays a key role in the postharvest pericarp browning of litchi fruit, but its underlying mechanism remains unclear. In the pericarp of newly-harvested litchi fruits, the LcPPO expression level was relatively high compared with developing fruits. Regardless of the litchi cultivar and treatment conditions, the LcPPO expression level and the PPO activity in pericarp of postharvest fruits exhibited the similar variations. When the fruits were stored at room temperature without packaging, all the pericarp browning index, PPO activity and the LcPPO expression level of litchi pericarps were reaching the highest in Nandaowuhe the most rapid browning cultivar , but the lowest in Ziniangxi the slowest browning cultivar within 2 d postharvest. Preserving the fruits of Feizixiao in 0.
Pericarp browning is the major post-harvest problem of litchi Litchi chinensis Sonn fruit, resulting in reduced commercial value of the fruit. Control of post-harvest pericarp browning of fruit using 9 different post-harvest treatments were studied. The treated fruits were packed in transparent perforated 0. On storage, pericarp browning increased irrespective of treatments with the decrease in pericarp specific activity, total pericarp phenol and total anthocyanin. Anthocyanin degradation index and polymeric colour increased during storage. National Center for Biotechnology Information , U. J Food Sci Technol.