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Summary Phytic acid acts as the major storage form of phosphorus in plant seeds and is poorly digested by monogastric animals. The degradation of phytic acid in animal diets is necessary to overcome both environmental and nutritional issues. The enzyme 1d‐myo‐inositol 3‐phosphate [Ins(3)P1] synthase (EC 5.5.1.4) catalyses the first step of myo‐inositol biosynthesis and directs phytic acid biosynthesis in seeds. The rice Ins(3)P1 synthase gene (RINO1) is highly expressed in developing seed embryos and in the aleurone layer, where phytic acid is synthesized and stored. In rice seeds, 18‐kDa oleosin (Ole18) is expressed in a seed‐specific manner, and its transcripts are restricted to the embryo and the aleurone layer. Therefore, to effectively suppress phytic acid biosynthesis, antisense RINO1 cDNA was expressed under the control of the Ole18 promoter, directing the same spatial pattern in seeds as RINO1 in transgenic rice plants. The generated transgenic rice plants showed strong ‘low phytic acid’ (lpa) phenotypes, in which seed phytic acid was reduced by 68% and free available phosphate was concomitantly increased. No negative effects on seed weight, germination or plant growth were observed. The available phosphate levels of the stable transgenic plants surpassed those of currently available rice lpa mutants.

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In most plant seeds, phosphorus is stored primarily as myo-inositol 1,2,3,4,5,6-hexakisphosphate (InsP 6 ; 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 phytic acid levels in rice seeds by manipulating the expression of the rice Ins(3)P 1 synthase gene RINO1 using transgenic methods. RINO1 cDNA was transformed into rice plants in the antisense orientation under the control of the rice major storage protein glutelin GluB-1 promoter. The T 4 generation of a stable transgenic line that contained four copies of the transgene showed little morphological differences compared to non-transgenic rice. In the T 5 seeds of this line, severe reductions in RINO1 protein levels were observed during the late maturing stages of ripening. Most of the T 5 seeds contained higher amounts of inorganic phosphates (Pi), without a reduction in total phosphorus levels, compared to non-transgenic seeds. Ion chromatography analysis suggested that the increase in available Pi is accompanied by a molar-equivalent decrease in phytic acid P. The expression patterns of RINO1 and GluB-1 were examined by quantitative real-time reverse transcriptase-polymerase chainreaction (RT-PCR). Potential strategies for further molecular breeding to reduce phytic acid levels in seeds are discussed.Keywords Gene expression AE Glutelin promoter AE myo-inositol 3-phosphate synthase AE Oryza sativa L. AE Phytic acid AE Transgenic plants Abbreviations DAF days after flowering Ins(3)P 1 1D-myo-inositol 3-phosphate InsP 6

The endosperm of cereal grains is an important resource for both food and feed. It contains three major types of tissue: starchy endosperm, the aleurone layer, and transfer cells. To improve grain quality and quantity using molecular methods, control of transgene expression directed by distinct temporal and spatial promoter activity is necessary. To identify aleurone layer-specific and/or transfer cell-specific promoters in rice, microarray analyses were performed, comparing the aleurone layer containing transfer cells and the other reproductive and vegetative tissues. After confirmation by RT-PCR analysis, we identified two putative aleurone layer and/or transfer cell-specific genes, AL1 and AL2. The promoter regions of these genes and β-glucuronidase (GUS) fusion constructs were stably transformed into rice. The GUS expression patterns indicated that the AL1 promoter was active exclusively in the dorsal aleurone layer adjacent to the main vascular bundle. In rice, transfer cells are differentiated in this region. Therefore, the promoter of the AL1 gene exhibits transfer cell-containing region-specific activity. The AL1 gene encodes a putative anthranilate N-hydroxycinnamoyl/benzoyltransferase. The promoter of this gene will be useful for enhancing uptake of nutrients from the mother cells and protecting filial seeds from pathogen attack.