Plant materials and growth conditions
Seed calli of rice (Oryza sativa L. cv. Nipponbare) were transformed with the use of Agrobacterium as described by Hiei et al. (1994). The generation of rice plants transgenic for OASA1D or OASA2, with both genes controlled by the promoter of the maize ubiquitin gene, has also been described (Tozawa et al., 2001; Yamada et al., 2004). Rice plants transgenic for the ß-glucuronidase gene (GUS) were also generated as described by Urushibara et al. (2001). All transgenic plants contained the hygromycin phosphotransferase gene (hpt) under the control of the 35S promoter of the cauliflower mosaic virus. Transgenic rice plants (R0 to R4) and non-transformed control plants (Nipponbare) were grown at 28 °C and 60% humidity under natural light conditions in pots (1/10 000a) containing podosol soil (Sumitomo Chemical, Osaka, Japan) in a containment greenhouse.
Two OASA1D transgenic lines, HW1 and HW5, were also grown in a non-containment greenhouse (R5) and an isolated field (R6). In 2002, seeds of the HW lines and Nipponbare were sown on 15 April and the seedlings were transferred individually to pots (1/10 000a) containing podosol soil on 15 May. The plants were maintained in the non-containment greenhouse under natural light and temperature conditions at the National Institue of Crop Science (NICS). No additional fertilizer was applied. In 2003, seeds of the HW lines and Nipponbare were sown on 14 May and the seedlings were transferred to an isolated paddy field (10 mx1.2 m) at the National Institute for Agro-Environmental Sciences in Tsukuba on 4 June. For each line, 40 seedlings were planted in duplicate in an area of 15 cmx30 cm per plant. The total amount of fertilizer applied m–2 included 6 g of N, 6 g of P2O5, and 6 g of K2O and was added at planting. Other practices followed the cultivation standards of Ibaraki Prefecture (Ibaraki Prefecture Standard Lowland Rice Cultivation, 1990).
Evaluation of agronomic traits
Agronomic traits, including heading date, culm length, and morphological characteristics, were evaluated by standard protocols (Ibaraki Prefecture Standard Lowland Rice Cultivation, 1990). Plants in the isolated field were harvested individually, air-dried, and analysed for yield and seed germination. All panicles were harvested from each plant, dried at 38 °C for 4 weeks, and then maintained in a freezer at –30 °C until analysis of amino acids and of IAA and metabolite profiling. For germination analysis, 15 or 20 seeds of individual lines were transferred to a filter paper that had been moistened with distilled water and placed in a Petri dish (6 cm in diameter); the analysis was performed in duplicate. The Petri dish was maintained at 35 °C for 15 d in the dark, and seed germination was assessed each day according to the modified standard method of NICS based on viviparity (Sakai et al., 2003b).
Amino acid analysis and determination of total nitrogen content
Dehulled seeds were autoclaved individually with 50 µl of water in a 1.5 ml Eppendorf tube for 15 min, and extracts were prepared from each seed as previously described (Wakasa and Widholm, 1987). The amounts of free amino acids were then quantified with the use of the PICO·TAG analysis system (Waters, Milford, MA). For quantitation of total Trp, five dehulled grains were pulverized with a mortar and pestle and heated in 5 M NaOH for 28 h at 110 °C; the hydrolysate was then acidified with 6 M HCl and subjected to analysis with an L-8800 High Speed Amino Acid Analyser (Hitachi High-Technologies, Tokyo, Japan).
Dehulled rice seeds were weighed and decomposed in concentrated sulphuric acid in the presence of salicylic acid and sodium thiosulphate. The ammonia formed was distilled, and was determined colorimetrically at 640 nm using the indophenol method.
Metabolite profiling
Four dehulled seeds were pulverized with a mortar and pestle and subjected to extraction for 1 h with 10 vols (v/w) of a mixture of water:methanol:acetic acid (249:250:1, by vol.). The extract was centrifuged at 16 000 g for 20 min, the resulting supernatant was passed through a SepPak C18 cartridge (Waters, Milford, MA), and the eluate was subjected to reversed-phase HPLC (LC-10Avp system; Shimadzu, Kyoto, Japan) with a Cadenza Column CD-C18 [250 mmx4.6 mm (inner diameter); Imtakt, Kyoto, Japan). Elution was performed with a mixture of acetonitrile and 0.02% aqueous trifluoroacetic acid (3:97 v/v, at 0 min; 30:70 v/v at 40 min; 98:2 v/v at 75 min) at a flow rate of 0.85 ml min–1 and a temperature of 40 °C; it was monitored with a photodiode array detector (Shimadzu SPD-M10Avp) over a wavelength range of 190–400 nm.
IAA analysis
Six dehulled seeds were pulverized with a mortar and pestle and then soaked for 3 h at 4 °C in 10 vol (v/w) of 80% acetone in water containing 2.5 mM diethyl dithiocarbamate. This extraction procedure was repeated three times. The combined extract was divided into three portions that were respectively subjected to quantification of free IAA, free plus ester forms of IAA and total IAA. The free IAA in the extract was partially purified by solid-phase extraction and quantified by liquid chomatography and tandem MS (LC-MS/MS) as previously described by Matsuda et al. (2005). For determination of the amount of free plus ester forms of IAA, the original extract was acidified to pH 2 with aqueous HCl and then analysed as for free IAA. For determination of the amount of total IAA, the extract was subjected to hydrolysis with 7 M NaOH for 3 h at 100 °C under N2 before analysis. [Phenyl-13C6]IAA was used as the internal standard in these analyses.
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