N6-methyladenosine on the natural antisense transcript of NIA1 stabilizes its mRNA to boost NO biosynthesis and modulate stomatal movement.

Nitric oxide (NO) is a crucial signaling molecule that regulates a wide range of metabolic pathways in different strata of organisms. In plants, nitrate reductase (NR) is a key enzyme for NO biosynthesis. There are two NR-encoding genes in Arabidopsis genome, NIA1 and NIA2, which are precisely regulated and expressed in a tissue-specific manner. In this study, we found that the natural antisense transcript as-NIA1, transcribed from the 3' UTR of NIA1, stabilizes NIA1 mRNA to maintain its circadian oscillation in plants grown under the light/dark cycle. Importantly, as-NIA1-dependent NIA1 mRNA stability is indispensable for NIA1-mediated NO biosynthesis in guard cells and natural stomatal closure. Moreover, we revealed that polypyrimidine tract-binding 3 (PTB3) regulates the stabilization of NIA1 mRNA by directly binding to UC-rich elements of as-NIA1. We further found that MTA deposits N⁶-methyladenosine (m⁶A) on as-NIA1, facilitating the as-NIA1-PTB3 interaction in vivo, in agreement with RNA structure prediction in that m⁶A-mediated structural alterations expose the UC-rich elements to enhance the accessibility of PTB3. Taken together, these findings reveal a novel molecular mechanism by which plants precisely manipulate NO biosynthesis to modulate light/dark-regulated stomatal movement, highlighting the coupling of RNA epigenetic modifications and structures shaping RNA-protein interactions in the regulation of hormone biosynthesis.