Two Young MicroRNAs Originating from Target Duplication Mediate Nitrogen Starvation Adaptation via Regulation of Glucosinolate Synthesis in Arabidopsis thaliana

Nitrogen is an essential macronutrient required for plant growth and development. A number of genes respond to nitrogen starvation conditions. However, the functions of most of these nitrogen starvation-responsive genes are unclear. Our recent survey suggested that many microRNAs (miRNAs) are responsive to nitrogen starvation in Arabidopsis thaliana. Here, we identified a new miRNA (miR5090) from the complementary transcript of the MIR826 gene. Further investigation uncovered that both miRNA genes recently evolved from the inverse duplication of their common target gene, ALKENYL HYDROXALKYL PRODUCING2 (AOP2). Similar to miR826, miR5090 is induced by nitrogen starvation. By contrast, the AOP2 transcript level was negatively correlated with miR826 and miR5090 under nitrogen starvation. GUS-fused AOP2 expression suggested that AOP2 was posttranscriptionally suppressed by miR826 and miR5090. miRNA transgenic plants with significantly low AOP2 expression accumulated fewer Met-derived glucosinolates, phenocopying the aop2 mutants. Most glucosinolate synthesis-associated genes were repressed under nitrogen starvation conditions. Furthermore, miRNA transgenic plants with less glucosinolate displayed enhanced tolerance to nitrogen starvation, including high biomass, more lateral roots, increased chlorophyll, and decreased anthocyanin. Meanwhile, nitrogen starvation-responsive genes were up-regulated in transgenic plants, implying improved nitrogen uptake activity. Our study reveals a mechanism by which Arabidopsis thaliana regulates the synthesis of glucosinolates to adapt to environmental changes in nitrogen availability.