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

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