Study on differentially expressed proteins during the fruit quality formation in loquat (Eriobotrya japonica)

Abstract: 【Objective】Loquat [Eriobotrya japonica (Thunb.) Lindl.] is a species of Eriobotrya plants belonging to Maloideae in family Rosaceae, which is an important fruit in Asia that ripens in late spring and early summer. Its fruit contains rich nutrients such as carotenoids, vitamins, minerals and amygdalin in flesh. It also has some health-beneficial effects, for example, expelling phlegm, arresting coughing, moistening lung, promoting appetite and helping digestion. Different regulation metabolisms in 3 different development periods (green fruit period, color changing period and yellow ripening period) were revealed during the fruit quality formation in loquat.【Methods】‘Jiefangzhong'loquat trees were planted in the National Germplasm Resources Nursery for Loquat in Fruit Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou, China. The fruits were harvested during 3 different fruit development periods from 7-year-old loquat trees. Total 27 fruits at each period were randomly divided into three groups, with each group having 9 fruits. The fruits were peeled and the pulps were immediately frozen in liquid nitrogen, and stored at-80 ℃ for further analysis. The proteins of loquat pulp were extracted using the Tris-buffered phenol method. The total proteins were separated by using two-dimensional gel electrophoresis (2-DE) with 24 cm, pH 4-7 linear immobilized pH gradient (IPG) strip in the first dimension, and after isoeletric focusing (IEF) , with 12% polyacrylamide gels in the second dimension (namely SDS-PAGE) . Differentially expressed protein spots with more than 2-fold differences in abundance at the three fruit development periods were found from the 2-DE gels by using the ImageMaster software. These differentially expressed proteins were identified using the matrix-assisted laser desorption/ionization tandem time-of-flight mass spectroscopy (MALDI-TOF/TOF-MS) and protein database search. The nutrient contents, including the total soluble sugar, reducing sugar, sucrose, carotenoid and ascorbic acid (AsA) , were measured by plant biochemistry methods at three fruit development periods. Meanwhile, the total RNA was extracted from the fruit pulp using the CTAB-LiCl method. The relative mRNA expression was analyzed using the RT-qPCR method in a CFX ConnectTM (BioRad) platform. The relative m RNA expression level of each gene was quantified using the 2-ΔΔCtmethod.【Results】To identify the proteins involved in the fruit development periods, 2-DE analysis was performed in 3 different development periods, in which more than 1 200 protein spots were detected for each period by using Image Master 2 D Platinum 5.0, respectively. Statistical analysis (p < 0.05) revealed a total of 56 protein spots with at least 2-fold differences in abundance as differentially expressed proteins in the green fruit period, color changing period, and yellow ripening period. Among them, 36 proteins were found to be up-regulated, 12 were down-regulated and the rest were up-regulated or down-regulated at the color changing period and yellow ripening period. These differentially expressed protein spots were excised from the gels, digested, and then analyzed by matching MALDI-TOFTOF/MS data to determine the biochemical characteristics of proteins. Fifty-five proteins were successfully identified, which were divided into 9 classes: sugar metabolism (16%) , tricarboxylic acid (TCA) cycle and energy metabolism (9%) , AsA-GSH cycle (14%) , carotenoid biosynthesis and ethylene formation (11%) , amino acid and protein synthesis (13%) , cell structure (8%) , defense stress (9%) , other regulatory proteins (13%) and so on, according to their physiological function. The proteins from sugar metabolism, TCA cycle energy metabolism and carotenoid biosynthesis and ethylene formation were upregulated, while the ones in the AsA-GSH cycle pathway were down-regulated. Visual and biochemical analysis in the fruit development of loquat revealed that the contents of total soluble sugar, reducing sugar, sucrose, carotenoid, and AsA increased with the fruit development, while the organic acid content decreased. To ascertain whether the protein expression levels were correlated with their mRNA contents, several genes encoding proteins related to carotenoids synthesis and ethylene formation metabolism (namely phytoene synthase (PSY) , chromoplaste beta lycopene cyclase (CYCB) , lycopene beta cyclase (LCYb) , lycopene epsilon cyclase (LCYe) , beta carotene ring hydroxylase (BCH) , zeaxanthin cyclase (ZEP) , 1-aminocyclopropane-1-carboxylate oxidase (ACO) ) were examined through quantitative RTPCR analyses. The result of RT-qPCR was similar to protein data. Compared with green fruit period, the relative mRNA expression levels of PSY, BCH, ZEP, LCYb, CYCB, and ACO were remarkably up-regulated in both color changing period and yellow ripening period.【Conclusion】The proteomic analysis of the loquat fruits at different development periods was performed in the present study. Fifty-five differential expressed protein spots were identified by MALDI-TOF/TOF-MS. The consistent results in the physiological analysis and relative mRNA expression level with the protein data reinforced each other to demonstrate an important scientific basis during the quality formation of loquat fruits. The quality formation of loquat fruits was closely related to these pathways including sugar metabolism, TCA cycle energy metabolism, carotenoid biosynthesis and ethylene formation.