Fig. 5

Quantitative real-time PCR analysis and in situ hybridization for OsASNase1 and OsASNase2 in flag leaves. The flag leaves were harvested during the ripening period from 14 to 35 days after flowering. The detection of transcript for OsASNase1 (filled square) and OsASNase2 (opened circle) were conducted in flag leaves using quantitative real-time PCR (a). Quantitative real-time PCR was performed using gene-specific primers for OsASNase1, OsASNase2 and actin, like described in Table 1. The relative contents of these transcripts were normalized against actin transcript. Means of four independent samples and standard error values (n = 3) are indicated. In situ detection of OsASNase2 transcript was conducted in flag leaves at 14 days after flowering (c and d). The antisense probe for OsASNase2 transcript was hybridized with the sections from flag leaves (c). The sense probe for OsASNase2 transcript was hybridized with the sections as negative control (d). The staining reaction was performed for 16 h. The structure of tissues was visualized with toluidine blue staining (b). Abbreviations: cc, companion cell; la, protoxylem lacuna; ms, mestome sheath cell; mc, mesophyll cell; ps, parenchyma sheath; se, sieve element and xv, xylem vessel element. Bars for (b) to (d) are 30 μm, respectively. Significant differences between OsASNase1 and OsASNase2 identified by Student’s t-test are marked with asterisks: *P < 0.05