nta-miR6019/6020 regulates <i>N</i> gene expression and function during seedling growth.

<p>(A) Maps of <i>N-CFP</i>^<i>T2T1</i> and <i>N-CFP</i>^<i>t2t1</i> constructs. The sequences of wild type and mutated miR6019/miR6020 binding sites are shown above and below the map. (B) Relative CFP transcript levels measured by RT-qPCR at 1-, 3- and 6 WAG in <i>N-CFP</i>^<i>T2T1</i> and <i>N-CFP</i>^<i>t2t1</i> transgenic plants. The <i>GAPDH</i> gene was used as the reference gene. CFP levels in 1 WAG <i>N-CFP</i>^<i>T2T1</i> is considered 1. (C) Secondary structures of wild type and AXC mutants of nta-MIR6019. Mature miRNA and miRNA star sequences are highlighted in red. (D) Northern blot hybridization of sRNAs isolated from <i>nta-MIR6019</i>^<i>WT</i><i>/NN</i> and <i>nta-MIR6019</i>^<i>AXC</i><i>/NN</i> transgenic plants and both TG34 and SR1 plants at 6 WAG. Small RNA sizes are indicated to the right. (E) Relative <i>N</i> transcript levels in <i>nta-MIR6019</i>^<i>WT</i><i>/NN</i> and <i>nta-MIR6019</i>^<i>AXC</i><i>/NN</i> plants, and both TG34 and SR1 seedlings at 6 WAG. The <i>GAPDH</i> gene was used as the reference gene. The lowest <i>N</i> transcript level in <i>N</i>-containing plants (<i>nta-MIR6019</i>^<i>WT</i><i>/NN-3</i>) is considered as 1. (F) <i>nta-MIR6019</i>^<i>WT</i><i>/NN</i> and <i>nta-MIR6019</i>^<i>AXC</i><i>/NN</i> transgenic plants and both TG34 and SR1 plants inoculated with TMV at 6 WAG. The total number (n) of test plants is indicated on the image. Bars = 1cm. The data are the means of three replicates with SD. Different letters indicate significant differences between the treatments according to one-way ANOVA test (P < 0.05). Y axes indicate relative fold change.