Fig. 3

Quantitative real-time PCR analysis and in situ hybridization for OsASNase1 and OsASNase2 in rice spikelets. The apical spikelets on the primary branches at the positions from the first (top) to the fifth were harvested during the ripening period from 7 to 35 days after flowering, respectively. The detection of transcript for OsASNase1 (filled square) and OsASNase2 (opened circle) was conducted in spikelets using quantitative real-time PCR (a). Quantitative real-time PCR analyses were 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 = 4) are indicated. Significant differences between OsASNase1 and OsASNase2 identified by Student’s t-test are marked with asterisks: ***P < 0.001. In situ detection of OsASNase2 transcript was conducted in spikelets at 14 days after flowering (c and d). The antisense probe for OsASNase2 transcript was hybridized with the sections from spikelets (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: al, aleurone layer; dvb, dorsal vascular bundle; ne, nucellar epidermis; np, nucellar projection; pp, phloem parenchyma cell; se, sieve element; xp, xylem parenchyma cell; and xv, xylem vessel element. Bars for (b) to (d) are 30 μm, respectively