The Role of bHLHm1 in Phytohormone Signalling in Legume Nodules and Roots

The GmbHLHm1 is a basic Helix-Loop-Helix membrane (bHLHm) DNA-binding transcription factor localized on the symbiosome cellular membrane. It regulates NH4+ transporters (AMFs) and nitrogen transporters (NRTs). RNAi-Silenced GmbHLHm1 transgenic hairy roots led to reduced nodulation, nitrogen fixation, and impaired plant growth. Overexpression of the GmbHLHm1 significantly induced nodulation, nitrogen fixation, , and enhanced plant growth. These results indicate that GmbHLHm1 plays an important role in nodulation and symbiotic nitrogen fixation. Long-term GA had a structural impact on the nodule, leading to a reduced expression of GmbHLHm1 and nitrogenase activity. GA application promoted shoot height on both the wildtype and RNAi- Silenced GmbHLHm1 transgenic hairy roots, indicating that induced shoot height is independent of GmbHLHm1 expression in roots. Extended IAA treatments induced GmbHLHm1 expression and enlarged nodules, while not changing the nodule biomass at the whole plant level. IAA treatment on GmbHLHm1-silenced nodules showed induced nodule size but not an increase in the nitrogen fixation area, indicating that auxin only improves the size of nodules, not the nitrogen fixation capability. The GmbHLHm1 promoter-GUS was mainly targeted to the infected cells and the nucleus of those cells. The GA treatment reduced the expression of the GmbHLHm1 in the infected region and repressed GmbHLHm1 expression in most of the outer layer cells. Yeast One-Hybrid experiments discovered five annotated proteins interact with the promoter of GmbHLHm1, and eleven for GmAMF3, including proteins related to both nodulation, membrane-localized proteins. With interrupted LjbHLHm1.1 expression in Lotus japonicus , plants had unhealthy nodule sand nitrogen deficiency symptoms, indicating the LjbHLHm1.1 expression plays an important role in L. japonicus growth and nodulation. L. japonicus plants and LjbHLHm1.1 mutant showed similar response to long-term GA treatment, as soybeans