Metabolism Mechanism Of Dietary Fibre Of Sweet Orang (Citrus Sinensis Osbeck)

Citrus fruits are rich in dietary fibre (DF), especially soluble dietary fibre (SDF) which is of value for human health. Thus, it has been focused to open out and utilize the DF of citrus. In this study, the concentration and composition of DF in the peel and pulp were comparatively analyzed among 'Cara cara' (Citrus sinensis Osbeck cv.), 'Newhall'(Citrus sinensis Osbeck cv.) and 'Tarocco Sanguine'(Citrus sinensis Osbeck cv.) orange during fruit development and maturation. Meanwhile, cell ultrastructure, mRNA transcript levels, subcellular localization and enzyme activity of hydrolases were analyzed. In addition, we have studied the effect of storage methods and pre-harvest foliar application of Ca, B on DF, to identify the relationship between gene expression of hydrolases and DF changes. Molecular, biochemical and ultrastructure characterization of the orange may provide new insights into the mechanism of DF metabolism. The main contents and results in the present study are as follows:(1) Changes of DF and hydrolases activities in development sweet orange fruitTotal dietary fiber (TDF), insoluble dietary fiber (IDF), SDF, cellulose (CEL), hemicellulose (HC), totale pectin (TP), protopectin, water soluble pectin (WSP) and polygalacturonase (PG), pectinesterase (PE), cellulase (Cx),β-galactosidase (β-Gdl) enzymes activities were determined in the peel and pulp of 'Cara cara', 'Newhall' navel orange and 'Tarocco Sanguine' at different developmental stages. The results showed that TDF contents in the peel and pulp of three sweet orange decreased gradually during the whole developmental period and the change tendency were identical in three varieties. 'Tarocco Sanguine' orange TDF contents throughout were least, 'Newhall' were next, 'Cara cara' most. The change tendency of IDF contents was accord with TDF, but against with SDF. Pectin contents decreased in fruit especially in pulp. WSP contents increased slighty during the later stage. Following growth of the fruit, the lignin and HC decreased rapidly. Lignin and HC contents were opposite correlation with Cx activities markedly. PG activities were lower in the fruit growth earlier period, along with the fruit mature increased quickly, and decreased rapidiy after the maximizing at the beginning of November. PE activities decreased in fruit especially in pulp, increased before fruit mature, and decreased along the fruit development in peel. The change tendency ofβ-Gal activities in pulp was accord with PE in peel, but there were no significant difference between green and mature fruit aboutβ-Gal activities in peel.(2) Effects of different storage method on DF and hydrolases activities of Cara cara navel orange fruit'Cara cara' fruit were stored in room as control. Pulp firmness, TDF, IDF, SDF, CEL, HC, TP, protopectin, WSP and PG, Cx, PE,β-Gal enzymes activities were determined in the peel and pulp of 'Cara cara' fruit stored on tree and in room at different storage stages. The results showed that contents of TDF, IDF, HC and CEL were diminished because of keeping fruits on tree, but WSP contents increased. As cell wall principal component HC, CEL hydrolyzed continuously and protopectin translated to WSP during storing on tree. It was negative correlation between Cx enzyme activity with CEL contents of stored 'Cara cara' fruit, Cx enzyme activity and the lignin contents of fruit storing on tree were negative correlation, too. There was no significant difference between fruit stored in room and on tree about PE andβ-Gal activities.(3) Effects of Ca, B on DF and hydrolases activities of Cara cara navel orange fruitIn the present study, specific activities of PG, Cx, PE,β-Gal were determined in order to identify the effect of pre-harvest application of Ca and B alone or in combination on DF of 'Cara cara' navel orange fruit. The results showed that the enzyme activities of PG, PE andβ-Gal were significantly reduced by pre-harvest application of Ca, B alone or in combination. Such treatments increased contents of TDF, IDF, CEL and protopectin, decreased SDF and WSP. Our study indicated that pre-harvest foliar application of Ca, B is useful for regulating the content of DF on 'Cara cara' navel orange.(4) Cloning, expression and subcellular localization of hydrolases gene in sweet orangeExpansin (Exp), PG, Cx, PE andβ-Gal were cloned from sweet arange, gene expression levels and subcellular localization of hydrolases were determined. The results showed that onion cells expressing the pC1302-Exp-GFP, pC1302-PE-GFP and pC1302-Gal-GFP showed cell wall GFP localization, when onion peels expressing pC1302-PE-GFP and pC1302-Gal-GFP were incubated on MS agar plates the pattern of fluorescence appeared different, they were located on the outer surface of the primary cell wall. The transcript levels of Exp increased following the fruit development. PG was largely responsible for pectin depolymerization and solubilization. The expression pattern of PE,β-Gal was similar in orange fruit, which showed a high level in the early stage of fruit development and decreased in the stage of fruit color turning, reaching a high level at the stage of fruit maturation. The transcript levels of Cx in orange fruit peel remained high in the green fruit stage and increased before fruit maturation in pulp. The results showed that the mRNA expression levels of PG increased significantly in the fruits of ST. Expression levels of PG, PE andβ-Gal were significantly reduced by pre-harvest application of Ca, B alone or in combination.(5) Changes of cell ultrastructure in the sweet orange fruitChanges of cell ultrastructure in the 'Cara cara' navel orange fruit segment membrane during maturation were assessed. The cells of early stage of fruit expansion were full and intact with different size of organelles and an obvious cell nucleus. The primary cell wall was very thick, and the cell middle lamella slightly apparent. At the late stage of fruit expansion, segment membrane cell were slightly different from those at the early stage. The middle lamella became distinct, and within cells, the cytoplasm was denser with a slight plasmolysis. At the early stage of fruit color turning, cell middle lamella became slightly thick and protoplasts in the cells became sparse. The cell plasmolysis was more apparent and cell organelles became faint with broken or uneven cell membranes. More significant changes in the cell ultrastructure occurred after the early stage of fruit color turning. The cell plasmalemma could not maintain a stable structure. The cell had large lumen, and the plasmalemma separated from the cell wall and shrank inwards. The middle lamella became distinct extremely and the primary cell wall became very thin. Following fruit maturation, the cell could not undergo the pressure, tortuosity and transmutation. Pre-harvest application of Ca, B had significant effect on the cross-linked polymer network of fruit segment membrane, the primary cell wall was very thick, the cell middle lamella was slightly apparent, and cell plasmalemma could maintain a stable structure.