miR169a-MhNF-YA2 module modulates H2S dosage-dependent lateral root formation via MhPIN1 and MhCYCB1;2 in Malus hupehensis.

Hydrogen sulfide (H2S) regulates lateral root (LR) formation dependent on its dosage, yet the underlying molecular mechanism remains not fully elucidated. Here, applying 0.5 mM and 0.05 mM NaHS promoted LR formation and upregulating miR169a expression in Malus hupehensis and Arabidopsis, respectively, whereas 1.0 mM NaHS inhibited that. Silencing miR169a reduced LR number and attenuated the improvement of 0.5 mM and 0.05 mM NaHS on LR formation, while its overexpression alleviated the 1.0 mM NaHS inhibition on LR formation. Furthermore, MhNF-YA2 was identified that directly targeted and inhibited by miR169a, and it functions oppositely to that of miR169a in regulating H2S dosage-mediated LR formation. Moreover, the MhNF-YA2 was found that directly repressed MhPIN1 and MhCYCB1;2 transcription under NaHS treatment. Overexpression of MhPIN1 and MhCYCB1;2 partially rescued the inhibitory effect of 1.0 mM NaHS on LR formation via increasing auxin level and meristem cell number in LRs, respectively. Conversely, suppression of MhPIN1 and MhCYCB1;2 in MhNF-YA2-suppression lines largely abolished the promotive effect of MhNF-YA2-suppression on LR formation, and this effect was alleviated by 0.5 mM NaHS but enhanced by 1.0 mM NaHS. Notably, all these regulatory processes were independent of H2S biosynthesis. Above all, our findings revealed that miR169a-MhNF-YA2 module mediates LR formation response to H2S dosage by regulating MhPIN1 and MhCYCB1;2 transcription, providing a new insight into the molecular mechanism by which H2S regulates LR formation.