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  • TC-I 2000 receptor br Materials and methods br Results and d


    Materials and methods
    Results and discussion
    Acknowledgements This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), and also by the project BioTecNorte (NORTE-01-0145-FEDER-000004) and the project MultiBiorefinery (POCI-01-0145-FEDER-016403) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte. SCS acknowledges her post doc grant (SFRH/BPD/88584/2012) from FCT. EA Macedo is a member of the Associate Laboratory LSRE-LCM, Project POCI-01-0145-FEDER-006984, funded by FEDER through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) – and by national funds through FCT – Fundação para a Ciência e a Tecnologia.
    Introduction Coconut (Cocos nucifera L.) is a monophyletic genus within the family Arecaceae (Palmae), a member of the subfamily Arecoideae that includes 27 genera and about 600 species of monocotyledonous flowering plants (Perera et al., 1998 & 2003). Coconuts have a diploid genome with 2n = 32 chromosomes and a haplophasic genome size (1C) of around 2.15 billion TC-I 2000 receptor pairs (Rohde et al., 2002). The coconut palm is called “tree of life” because it provides almost all the necessities of life including food, drink, oil, medicine, fiber, timber, thatch, and other domestic utensils (Foale, 2003). Curd coconut is an abnormal endosperm phenotype, often found in tall type coconuts (Santoso et al., 1996 and Islam et al., 2009). In normal plantation, only few curd coconut fruits occur only from some coconut plants. Unlike normal coconut, the solid endosperm of curd coconut becomes soft, while its liquid endosperm becomes thick or filling the entire cavity as a soft gel (Islam et al., 2009), as shown in Fig. 1. The curd coconut is in high demand due to its soft fatty and delicious texture and the price of a fruit is about 10 times of a normal coconut (Wattanayothin, 2004, 2005). In addition, the phenotype of curd coconut was found to be a pure genetic factor of a single gene locus, which is inherited in autosomal recessive fashion (Zuniga, 1953). Galactomannans make up to 61% of the total carbohydrate content of a mature normal coconut kernel (galactose to mannose ratio 1:12); and they play a structural role in the formation of the secondary cell wall in coconut solid endosperm (Balasubramaniam, 1976). Whereas in curd coconut kernel, cell wall was principally 60% water soluble galactomannan with galactose to mannose ratio of 1:2 (Flavier, 1999). In normal coconut or other seed plants, cell wall storage polysaccharides are degraded and mobilized as sucrose, glucose or fructose to supply nutrient during seed germination (Buckeridge, 2010). While the curd coconut contains an apparently normal embryo, it fails to germinate in vivo because its endosperm is incapable of supporting embryo germination. However, germination of curd coconuts is successful in vitro using tissue culture techniques (De Guzman and Del Rosario, 1964; Ashburner et al., 1995). Galactomannans, a component of the cell wall, consist of a linear backbone of β-(1 → 4)-linked D-mannose residues to which D-galactose residues are attached by α-(1 → 6) linkages. The degradation of galactomannan requires the presence of three enzymes working together: namely endo-β-mannanase (EC, β-mannosidase (EC and α-galactosidase (EC (Reid and Meier, 1973). Each of these enzymes have specific mechanisms for cleaving sugar residues at different sites: endo-β-mannanase randomly cleaves endo (1 → 4)-β-D-mannosidic bonds while β-mannosidase removes terminal (1 → 4)-β-D mannosides from the ends of the oligogalactomannan, and α-galactosidase cleaves the (1 → 6)-α-D galactoside residues from the non-reducing end of oligogalactomannan. Investigations into the comparative activities of these three enzymes within the endosperms of both normal and curd coconuts reveal that in normal coconut, only α-galactosidase activity increased continually with endosperm maturation, and was inversely correlated with the amount of galactomannan. In contrast, only α-galactosidase activity could not be detected in any stage of curd coconut endosperm development except the very last stage (11–12 months of age) where its activity was 8300 folds lower than that of the normal coconut (Mujer et al., 1984; Samonte et al., 1989). Thus, these studies confirmed lacking of endosperm specific coconut α-galactosidase (CnAGal) plays a major role in the in vivo degradation of oligogalactomannan in the endosperm. In addition, the deficiency of CnAGal activity affects the accumulation of oligogalactomannan, which causes curd coconut endosperm (Mujer et al., 1984). However, neither the cDNA encoding CnAGal gene nor mutations in the gene has been identified and characterized. This study therefore aims to isolate and characterize the wild-type α-galactosidase gene from normal coconut, and identify the types of mutation presenting in the α-galactosidase gene, resulting in the curd coconut phenotype.