Differences Among Litchi Pericarp Browning Caused By Different Stress Treatments And Its Mechanisms

Litchi(Litchi chinensis Sonn.), a typical tropical and subtropical fruit, is native to South China with extremely important role in the south China rural economy. Litchi fruit is very easy to turn brown after harvest because of the high temperature and high humidity. In consequence, it limits the sales, storage and transportation. Exploring the browning mechanisms after harvest on litchi has guiding significance to develop the technologies on litchi preservation. There are many factors that can lead to browning such as dehydration, high and low temperature damage, chemical damage, microbial infection, and so on. So far, the discrepancies of pericarp browning induced by diverse stress factors is not clear yet, let alone the mechanism of browning due to different types stress factors.In this paper, ’Feizixiao’ and ’Nuomici’ litchi fruit were used as materials. After treated with dehydration(unpacked), hot water(60?C,2min), short-time freezing(-20?C,1h), hydrochloric acid(6%edible hydrochloric acid) and inoculate pathogen(Peronophythora litchi) stress, litchis were stored at ambient temperature toinvestigate the appearance, ultra-structure, physiological and biochemical properties, genetic expression and proteomics in pericarp, and the main results are as follows:(1)Though certain difference are presented in Changes in pericarp color among different stress treatments, all fruits bacame brown quickly after treatment and the browning index came to 5 at the later storage. As the browning aggravate gradually, L*, a* and C* all show a gradual decrement trend, with a slight increase in h?. The speed rate and representation in browning of litchi pericarp are vary from different stress factors. The browningspreads evenly in dehydration treatment litchi fruit, presenting dried yellow; the lowest L* and a* appeared at the hot water treated litchi that with the highest h0, the totally browning pericarp are dark chocolate-brown; the short-time freezing treated fruit show up the supreme L* and a*. For five types of browning, the browning index of pericarp is significantly related to L*, a* and C*, therefore, we could measure the degree of browning on the basis of L*, a* and C*.(2)By comparing and analyzing theultra-structure of fresh and browning litchi pericarp after different stress treatment, we find that the cellular structure of fresh fruit pericarp remained intact under SEM(scanning electron microscope) and TEM(transmission electron microscope) observation, and the cells remained alive well, meanwhile contains abundant organelles. Five stress treatments have all destroyed the cellular structure, losing the cell vitality, but there are differences among five types of browning. Litchi pericarp structure was damaged thoroughly after browning due to water stress treatment, accompanied by fracture of the cell walls and falling away of the cellular contents; cells of browning pericarp due to hot water treatment became shrinkage with most endocyte left; after short-time freezing treatment, the cellular structure of browning pericarp is relatively complete with few contents; wax layers falls off seriously after treated with hydrochloric acid; for inoculated ’Feizixiao’ litchi fruit, there are millions of particles in browning pericarp cells, but next to nothing in the cells of ’Nuomici’ litchi fruit.(3)Certain differences are presented in Changes in the physiological and biochemical index among five stress treatment. The respiration rate of DT, HWT and SFTfruits declined gradually when the ethylene evolution shows small fluctuation; HAT and IT have motivated the respiration rate and ethylene evolution especially the later stage of browning. Lac, PPO, POD and SOD enzymatic activity were supressed after HWT in ’Feizixiao’ litchi fruit, but not the same in ’Nuomici’ fruit. In two cultivar litchi, Lac and PPO enzymatic activity were lower in HAT fruit which with higher POD and SOD enzymatic activity. During storage, water and anthocyanin content in pericarp of five treatment litchis degreased whereas the anthocyanin content increased slightly at the later stageof storage with HAT. Content of total phenols and flavonoids increased in pericarp of HAT fruits, but decreased in other four groups;however, the above two show a rising trend in all ’Nuomici’ litchi. In pericarp of HWT, SFT and HAT fruit, the increasing trend of MDA and proline content was restrained.(4)There is diversity in relative expression of Lc Lac, Lc PPO, POD and SODafter treated with different stress treatment. During the process of browning, the relative expression level of Lc Lac, POD and SOD were all nethermore except the Lc PPO. The relative expression of Lc Lac raised at first, then decreased in DT, HAT and IT fruits while there is a sustained downward trend in HWT fruits. The changed trend of Lc Lac expression was rise-fall pattern in ’Feizixiao’, but fall-rise pattern in ’Nuomici’. As the browning get deepen, the relative expression of Lc PPO rose first and fell later in DT fruits while increased gradually in other four treatments. The relative expression of four POD genes was low in two cultivars of litchi, and fluctuated smoothly in some ranges. The expression of POD5 was enhanced while the POD3 was inhibited in HWT fruits; after treated with hydrochloric acid, the POD3 and POD5 gene expression were all accelerated. The trends of Fe-SOD gene expression was sustained downward in DT, HWT and IT fruits, while steady in SFT and HAT fruits. The expression of Mn-SOD gene was inhibited to a certain degree in HWT, SFT and HAT fruits. Hot water treatment stimulated the expression of Cu/Zn-SOD-1, Cu/Zn-SOD-2 å'Œ Cu/Zn-SOD-3, but hydrochloric acid and inoculatation treatment had little impact on the gene expression.(5) Correlational and stepwise regression analysis showed thatthe main factors to browning in five stress treatment were distinct. For HAT and IT fruits, the browning index was correlated significantly with water content which was the main factor to the two types of browning. For other treatments, the main factors to browning depend on the cultivar of litchi. Content of anthocyanin, total phenols, flavonoids, MDA,proline and water content weresignificantly correlated with the browning index in DT fruits, the main factors to browning were Lc Lac gene expression in ’Feizixiao’ while water content, PPO enzymatic activity and Lc PPO gene expression in ’Nuomici’. For HWT fruits, the water content,content of flavonoids and Lc Lac gene expression were significantly correlated with the browning index, and the main factors to browning were SOD enzymatic activity in ’Feizixiao’ while POD3 gene expression and water content in ’Nuomici’. Content of total phenols, proline, water content and Lc PPO gene expression were significantly correlated with the browning index in SFT fruits, the main factors to browning were water content and Cu/Zn-SOD-2 gene expression in ’Feizixiao’ while content of anthocyanin and Cu/Zn-SOD-2 gene expression in ’Nuomici’.(6) Proteomics were used to analyze and discuss the proteomic Changes in different browning degree of litchi pericarp. By combining the TMT labels and mass spectrometry, we got 10,793 secondary spectrums that can be matched in the database and 6607 specific peptides, in which 1957 proteins can be used for quantitative analysis. Through a comparative analysis with three browning pericarp of different degree, we obtained 603 differentially expressed proteins in which 10 of them are in common. The prediction functions of 10 proteins are concentrated on metabolic process, gene expression, biological process, response to stress and stimulus, cellular protein metabolic process, carbohydrate metabolic process and so on. Considering the significant differently expression and labeling informations, we select the arabinofuranosidase(Ara) and heat shock protein 70(Hsp70)for subsequent analysis. We cloned the two gene c DNA fragments corresponding the two protein, and accomplished the quantitative analysis. Along with the pericarp browning, the expression of Ara protein and gene were all increased gradually, but theexpression of Hsp70 protein first rose then fell whiletheexpression of Hsp70 gene continued to rise. It is supposed that Ara protein and gene are likely to be involved in the browning of litchi pericarp.