| Research Abstract |
In most plant seeds, phosphorus is stored primarily as myo-inositol 1,2,3,4,5,6-hexakisphosphate (InsP_6), or phytic acid. Reducing the phytic acid content of seeds is a major breeding target both to increase the availability of mineral nutrients and to decrease the environmental load of phosphorus. The first step in phytic acid biosynthesis and inositol metabolism is catalyzed by 1D-myo-inositol 3-phosphate (Ins(3)P_1) synthase. In this study, we aimed to reduce the phytic acid levels in rice seeds by manipulating the expression of the rice Ins(3)p_1 synthase gene RINO1 using transgenic methods. RINO1 was transformed into rice plants in the antisense orientation under the control of the promoter of the glutelin gene GIuB-1, which encodes a seed storage protein and shows high and seed-specific expression during rice seed maturation. The T_4 generation of a stable transgenic line that contained four copies of the transgene showed no differences in morphology compared to non-transgenic p
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lants. The T_5 seeds of this line contained less RINO1 protein than did non-transgenic seeds during the late stages of ripening. Most of the T_5 seeds contained greater amounts of inorganic phosphates (Pi) than did non-transgenic seeds. Pi concentrations in non-transgenic and transgenic seeds were 2.77% and 17.29%, respectively. The expression patterns of RINO1 and GluB-1 were examined by quantitative real-time reverse transcriptase-polymerise chain-reaction (RT-PCR). These results suggested that higher effects could be obtained by using a promoter with the same temporal and spatial activity in the seeds as RINO1, thereby reducing the levels of RINO1 protein earlier in the process of seed maturation.
Despite many physiological investigations of phytic acid accumulation and storage, little is known at the molecular level about the biosynthetic pathway in plants. Recent work has suggested two pathways for InsP_6 synthesis. One is an inositol lipid-independent pathway that occurs through the sequential phosphorylation of Ins(3)P_1. The other is a phosphoipase C-mediated inositol lipid-dependent pathway in which inositol 1,4,5-trisphosphate (Ins(1,4,5)P_3) is sequentially phosphorylated to InsP_6. We identified 12 genes from rice that code for the enzymes that may be involved in the metabolism of inositol phosphates. These enzymes include Ins(3)P_1 synthase, inositol monophosphatase, Ins(1,4,5)P_3 kinase, inositol 1,3,4,5,6-pentakisphosphate 2-kinase (Ipk1), and inositol 1,3,4-teiskisphosphate 5/6-knase (ITP5/6K). The quantification of absolute amounts of mRNAs by real-time RT-PCR revealed the unique expression patterns of these genes. We showed that transcripts of RINO1, Ipk1, two genes out of six ITP5/6K are highly expressed in developing embryos, where vigorous synthesis of phytic acid occurs. The expression level was much lower in other organs This overexpression is consistent with the idea that these enzymes are needed to maintain the high level of physic acid synthesis in developing rice seeds. The results here also suggested that the inositol lipid-independent pathway is a major route of physic acid biosynthesis in rice seeds. Less
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