No abstract

The amyloplast, a form of differentiated plastid, proliferates in sink tissues, where it synthesizes and stores starch granules. Little is known about the molecular mechanism for amyloplast division and development. The rice (Oryza sativa) endosperm provides an excellent model system for studying molecular mechanisms involved in amyloplast division and starch synthesis. We compared amyloplast division processes in the endosperm of wild type and a mutant of ARC5, a member of the dynamin superfamily. Plant growth and fertility of arc5 were not significantly different from the wild type. Unlike binary fission of chloroplast in the leaf, small amyloplasts in the endosperm of wild type divide simultaneously at multiple sites, generating a beads-on-a-string structure. In addition, large amyloplasts divide by budding-type division, giving rise to small amyloplasts attached to their surfaces. ARC5 and FtsZ2-1 fused to fluorescent proteins were targeted to the constriction sites in dividing amyloplasts. Both the loss of function of ARC5 and overexpression of ARC5 fusion proteins in the endosperm did not produce spherical amyloplasts with increased diameter, but produced either fused amyloplasts with thick connections or pleomorphic types, suggesting that proper stoichiometry between ARC5 and other components in the amyloplast division machinery is necessary for the completion of the late stage of amyloplast division. The size distribution of starch granules purified from arc5 was shifted to small and the starch gelatinization peak temperature was significantly higher than for wild-type starch, suggesting that amyloplast division processes have a significant effect on starch synthesis.

Storage tissues such as seed endosperm and tubers store starch in the form of granules in the amyloplast. In the rice (Oryza sativa) endosperm, each amyloplast produces compound granules consisting of several dozen polyhedral, sharp-edged and easily separable granules; whereas in other cereals, including wheat (Triticum aestivum), barley (Hordeum vulgare) and maize (Zea mays), each amyloplast synthesizes one granule. Despite extensive studies on mutants of starch synthesis in cereals, the molecular mechanisms involved in compound granule synthesis in rice have remained elusive. In this study, we expressed green fluorescent protein (GFP) fused to rice Brittle1 (BT1), an inner envelope membrane protein, to characterize dividing amyloplasts in the rice endosperm. Confocal microscopic analyses revealed that a septum-like structure, or cross-wall, containing BT1-GFP divides granules in the amyloplast. Plastid division proteins including FtsZ, Min and PDV2 play significant roles not only in amyloplast division, but also in septum synthesis, suggesting that amyloplast division and septum synthesis are related processes that share common factors. We propose that successive septum syntheses which create sections inside the amyloplast and de novo granule synthesis in each section are primarily responsible for the synthesis of compound granules.

Debranching enzymes, which hydrolyze α-1 and 6-glucosidic linkages in α-polyglucans, play a dual role in the synthesis and degradation of starch in plants. A transposon-inserted rice mutant of isoamylase3 (isa3) contained an increased amount of starch in the leaf blade at the end of the night, indicating that ISA3 plays a role in the degradation of transitory starch during the night. An epitope-tagged ISA3 expressed in Escherichia coli exhibited hydrolytic activity on β-limit dextrin and amylopectin. We investigated whether ISA3 plays a role in amyloplast development and starch metabolism in the developing endosperm. ISA3–green fluorescent protein (GFP) fusion protein expressed under the control of the rice ISA3 promoter was targeted to the amyloplast stroma in the endosperm. Overexpression of ISA3 in the sugary1 mutant, which is deficient in ISA1 activity, did not convert water-soluble phytoglycogen to starch granules, indicating that ISA1 and ISA3 are not functionally redundant. Both overexpression and loss of function of ISA3 in the endosperm generated pleomorphic amyloplasts and starch granules. Furthermore, chloroplasts in the leaf blade of isa3 seedlings were large and pleomorphic. These results suggest that ISA3 facilitates starch metabolism and affects morphological characteristics of plastids in rice.