Cloning and expression analysis of litchi (Litchi Chinensis Sonn.) polyphenol oxidase gene and relationship with postharvest pericarp browning

Abstract

Polyphenol oxidase (PPO) plays a key role in the postharvest pericarp browning of litchi fruit, but its underlying mechanism remains unclear. In this study, we cloned the litchi PPO gene (LcPPO, JF926153), and described its expression patterns. The LcPPO cDNA sequence was 2120 bps in length with an open reading frame (ORF) of 1800 bps. The ORF encoded a polypeptide with 599 amino acid residues, sharing high similarities with other plant PPO. The DNA sequence of the ORF contained a 215-bp intron. After carrying out quantitative RT-PCR, we proved that the LcPPO expression was tissue-specific, exhibiting the highest level in the flower and leaf. 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 0.2-μm plastic bag at room temperature would decrease the rate of pericarp water loss, delay the pericarp browning, and also cause the reduction of the pericarp PPO activity and LcPPO expression level within 3 d postharvest. In addition, postharvest storage of Feizixiao fruit stored at 4°C delayed the pericarp browning while decreasing the pericarp PPO activity and LcPPO expression level within 2 d after harvest. 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.

Figure 1. Phylogenetic tree of LcPPO with other PPOs from different species.

All of PPO sequences except LcPPO (JF926153 Litchi chinensis) were from NCBI database. 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 1000 replicates.

Figure 2. Southern blotting analysis of LcPPO.

Numbers on the top of the lane represent that Genomic DNA from Feizixizo leaves was digested by EcoR I (1), and Xho I and EcoR I (2) at first, and then was hybridized with the LcPPO-specific probe. A clear band was visible in lane 1 and lane 2.

Figure 3. Expression of LcPPO in different litchi tissues of Feizixiao.

The different letter above the column indicated that there were significantly different among the samples (P<0.05). The highest expression was found in the flower and leaf, and the lowest expression was in the pulp.

Figure 4. LcPPO expression in litchi pericarp at different developmental stages.

Fruits of Feizixiao were harvested on May 14^th, 2010; Ziniangxi and Nandaowuhe were harvested on July 7^th, 2010. Expression of LcPPO in pericarp increased sharply after harvested. Similar changes were observed with Feizixiao, Ziniangxi and Nandaowuhe.

Figure 5. Changes in pericarp-browning index, PPO activity, and LcPPO expression of postharvest litchi fruits of different cultivars.

Pericarp-browning index (A) of Nandaowuhe increased fast compared with Feizixiao and Ziniangxi. PPO activity (B) for Nandaowuhe was higher than Feizixiao and Ziniangxi, but LcPPO expression (C) level increased fast than others on the first day after harvest, and then both Nandaowuhe and Feizixiao reached highest level on day 2.

Figure 6. Changes in pericarp-browning index, pericarp water loss, PPO activity, and LcPPO expression of Feizixiao fruit under packaging treatment.

A. Pericarp-browning index: B. Pericarp water loss; C. PPO activity; and D. LcPPO expression. Fruit bagging reduced the water loss in pericarp of Fizixiao, and slowed down the pericarp-browning. During the first three days of storage, both the PPO activity and the LcPPO expression were decreased in bagged fruit.

Figure 7. Changes in pericarp-browning index, PPO activity, and LcPPO expression of Feizixiao fruit after stored at the low temperature.

A. Pericarp-browning index: B. PPO activity, and C. LcPPO expression Pericarp browning was delayed while stored the fruit at 4°C. The low temperature also can cause the reduction of the PPO activity and LcPPO expression level in pericarp during the first 2 days after harvest.