Figure 2. Predicted target OsaSULTR1 and OsaSULTR2 exhibit opposite expression patterns to that of the OsamiR395 in rice root.

(a) Target sites of the four putative OsamiR395 target genes in rice. The target sites were compared with the complementary sequence of mature OsamiR395h. Asterisks indicate the identical sequences. (b) RT-PCR analysis of expression levels of the OsamiR395 putative targets. Total RNA samples used for RT-PCR were extracted from leaf and root tissues of two weeks old rice grown in N6 medium with 0, 20, 1500 or 2000 μM (NH⁴⁺)₂SO₄ and used for RT-PCR analysis. OsaSIZ1 was used as a reference gene. Experiment was repeated three times. (c) Stem-loop real-time RT-PCR analysis of mature OsamiR395 and real-time RT-PCR analysis of pri-OsamiR395h. Total RNA samples were prepared from leaf and root tissues of two weeks old rice grown in regular N6 medium (+S) or N6 medium without SO₄⁺ (−S) and used for RT-PCR analysis. OsaSIZ1 was used as a reference gene. (d) Real-time RT-PCR analysis was also conducted to determine the expression levels of the OsamiR395 putative targets in rice leaves and roots. Total RNA samples were prepared as in (c) and used for real-time RT-PCR analysis. OsaSIZ1 was used as a reference gene. For (c,d), data are presented as means of two independent biological replicates and three technical replicates, error bars represent SD (n = 6). Asterisks indicate the significant differences between expression levels under −S and +S conditions. P < 0.05 is marked as*, P < 0.01 is marked as**.