Abstract
Rice (Oryza sativa) endosperm has two isoamylase (ISA) oligomers, ISA1 homo-oligomer and ISA1-ISA2 hetero-oligomer. To examine their contribution to starch synthesis, expression of the ISA1 or ISA2 gene was differently regulated in various transgenic plants. Although suppression of ISA2 gene expression caused the endosperm to have only the homo-oligomer, no significant effects were detected on the starch phenotypes. In contrast, ISA2 overexpression led to endosperm having only the hetero-oligomer, and starch synthesis in the endosperm was drastically impaired, both quantitatively and qualitatively, because the starch was devoid of typical starch features, such as thermal and x-ray diffraction properties, and water-soluble highly branched maltodextrins were accumulated. In the ISA2 overexpressed line, about 60% to 70% of the ISA1-ISA2 hetero-oligomer was bound to starch, while the ISA homo- and hetero-oligomers from the wild type were mostly present in the soluble form at the early milking stage of the endosperm. Detailed analysis of the relative amounts of homo- and hetero-oligomers in various lines also led us to the conclusion that the ISA1 homo-oligomer is essential, but not the ISA1-ISA2 oligomer, for starch production in rice endosperm. The relative amounts of ISA1 and ISA2 proteins were shown to determine the ratio of both oligomers and the stoichiometry of both ISAs in the hetero-oligomer. It was noted when compared with the homo-oligomer that all the hetero-oligomers from rice endosperm and leaf and potato (Solanum tuberosum) tuber were much more stable at 40°C. This study provides substantial data on the structural and functional diversity of ISA oligomers between plant tissues and species.
Highlights
Rice (Oryza sativa) endosperm has two isoamylase (ISA) oligomers, ISA1 homo-oligomer and ISA1-ISA2 hetero-oligomer
The mechanism by which debranching enzyme (DBE) are involved in the synthesis of amylopectin is still debatable, it is thought that these enzymes play a crucial role in amylopectin biosynthesis by removing excess branches (Ball et al, 1996) or improper branches (Nakamura, 2002) that interfere with the formation of double helices of the cluster chains of amylopectin (Nakamura, 2002) and crystallization of the starch (Myers et al, 2000)
The results show that the ISA1 homo-oligomer is indispensable for amylopectin biosynthesis in rice endosperm, while the ISA1-ISA2 hetero-oligomer seems to be essential for starch synthesis in leaf, it is insufficient for normal starch accumulation in the endosperm
Summary
Rice (Oryza sativa) endosperm has two isoamylase (ISA) oligomers, ISA1 homo-oligomer and ISA1-ISA2 hetero-oligomer. The loss of ISA1 protein in these plants results in a tremendous obstacle in the formation of starch granules in cereal endosperms such as maize (Zea mays; James et al, 1995), rice (Oryza sativa; Kubo et al, 1999), and barley (Hordeum vulgare; Burton et al, 2002), Arabidopsis (Arabidopsis thaliana) leaves (Delatte et al, 2005; Wattebled et al, 2005), potato (Solanum tuberosum) tuber (Hussain et al, 2003), and Chamydomonas cells (Mouille et al, 1996) In these tissues or cells, starch was observed to be replaced by a sort of water-soluble highly and randomly branched polysaccharide devoid of the cluster structure called phytoglycogen. The mechanism by which DBEs are involved in the synthesis of amylopectin is still debatable, it is thought that these enzymes play a crucial role in amylopectin biosynthesis by removing excess branches (Ball et al, 1996) or improper branches (Nakamura, 2002) that interfere with the formation of double helices of the cluster chains of amylopectin (Nakamura, 2002) and crystallization of the starch (Myers et al, 2000)
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