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We used the rice variety Oryza sativa L. ‘ … We used the rice variety Oryza sativa L. ‘Nipponbare’. Homozygous lines of OsGS1;1 knockout mutants were generated by inserting the endogenous retrotransposon Tos17 (Hirochika, 2001) into the OsGS1;1 gene. We selected the NC2373 and ND8037 lines, which carried a Tos17 insertion only in the OsGS1;1 open reading frame (Tabuchi et al., 2005). The knockout lines exhibited very low fertility and inhibited grain filling; therefore, heterozygous seeds were used to obtain homozygous plants. Wild type and two independent heterozygous lines were grown as described previously (Yamaya et al.,1995). Briefly, in May 2010, July 2008 and September 2005 and 2008,WT and heterozygous seeds were soaked in water (Dataset S1) at 30°C for 36–48 h under dark conditions and the germinated seeds were transferred to a nylon net that floated on culture medium in a plastic container. When the endosperm had been fully utilised,seedlings were transferred to different nutrition conditions to measure different N availabilities. The WT and heterozygous plants were cultivated in a greenhouse with the following conditions for metabotyping in June (14 h of light at 26°C and 10 h of darkness at 23°C; without controlling humidity; light strength, 810 μmol m-2 sec-1), while we used another greenhouse for other experiments (12 h of light at 30°C and 12 h of darkness at 25°C; without control humidity). To identify homozygous mutants, the second LB of each plant was harvested for PCR analysis. each plant was harvested for PCR analysis.
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