MAP-Based Cloning And Functional Analysis Of A Key Gene FLO6 Involved In Regulation Of Starch Synthesis In Rice Endosperm

Starch is the most widespread form of energy storage in the plant kingdom. Although many enzymes and regulators have been identified for starch biosynthesis, new players remain to be identified given that it is a complicated and sophisticated process. On the other hand, starch, as main component of rice endosperm, the content and physicochemical properties of stach directly affect the cooking qulity of rice. Meanwhile, the accumulation of starch in rice endosperm also affects the rice yield. Therefore, it is a meaningful and significative work to study the pathway of starch biosynthesis with rice as a model plant.The abnormal endosperm mutant of rice is an agronomical trait that has been used for the study of starch synthesis, such as waxy, dull, sugary, shrunk and floury. In this work, we investigated the underlying mechanism of a novel floury mutant-flo6 (floury endosperm 6). Map-based cloning showed that FLO6 encodes a 529aa protein of unknown function. The FLO6 protein harbors an N-terminal transit peptide that ensure its correct localization and function in the plastid, and a C-terminal CBM48 (carbohydrate-binding module48) domain that binds to starch. In addition, FLO6 interacts with ISA1 but not influence ISA1 activity. These data provided a basis to clarify the starch biosynthesis pathway in rice endosperm. The main results are as follows:1. We report the isolation and characterization of a new rice floury mutant named flo6 (come from cultured tissue of the japonica type rice cv. Nipponbare), which is required for starch synthesis in rice endosperm. On one hand,the flo6 mutant showed a markedly slower grain-filling rate and lower starch content throughout endosperm development. On the other hand, the pasting property, swelling power of starch, as well as the stucture of amylopectin in flo6 were changed, resulted in different physicochemical properties of starches in flo6 seeds. It is, therefore, likely that genetic modification of the gene for FL06 will lead to the synthesis of novel types of starch. Thus, starch synthesis in flo6 mutant endosperm is severely disrupted both quantitatively and qualitatively.2. Our results show that FLO6 plays important roles in compound granule formation in rice. Compare to wild-type, smaller scattered SGs in the cytosol and three types of abnormal structure of compound granules were observed in the flo6 endosperm cells suggesting that FLO6 is required for maintaining the structure of compound granule in rice.3. A Map-based cloning approach was used to isolate the FLO6 gene. We first crossed theflo6 mutant with an indica varity 9311 to generate a F2 mapping population and the flo6 locus was mapped to a 26kb genomic region flanked by the marker PC-12 and PC-11. Sequece analysis revealed a single nueotide insertion in Os03g0686900, which led to a premature stop codon. Futher analysis showed that the expression of Os03g0686900 was downregulated in flo6 mutant. Function complementation analysis demonstrated Os03g0686900 correspond to the candidate FLO6 gene. The FLO6 gene encodes a 529aa protein of unknown function. The FLO6 protein harbors an N-terminal transit peptide that ensure its correct localization and function in the plastid, and a C-terminal CBM48 (carbohydrate-binding module48) domain that binds to starch.4. Yeast two-hybrid assay showed that FLO6 can interact with isoamylasel (ISA1) but not any starch synthesis enzyme. Interaction relationship between FLO6 and ISA1 was further confirmed by way in vitro pull-down and in vivo BiFC experiment. In addition, Zymogram analyses showed that no significant differences in the activities of DBE isozymes (isoamylase and pullulanase) including ISA1 homo-oligomer and ISA1-ISA2 hetero-oligomer, SS isoforms (SSI and SSIIIa), PHO isoforms (PHO1 and PHO2) and BE isoforms (BEI, BEIIa, and BEIIb), were found between the flo6 mutants and the wild-type, indicating that the mutation of FLO6 has no effect on the activity of the main starch biosynthesis enzymes. In our starch pull-down array showed that ISA1 does not bind to starch directly. However ISA1 can be detected in starch granule-bound fraction in vivo. These results indicate that FLO6 might act as a scaffold protein which interacts with ISA1 and regulates the binding of ISA1 to starch.