# Identification of symbiotic nitrogen fixation‐modulating factors in alfalfa and mechanism elucidation of MsHHO3

## TL;DR

This study identifies MsHHO3 as a key regulator of nitrogen fixation in alfalfa, showing how it controls nodule formation and nitrogenase activity in response to nitrate.

## Contribution

The paper reveals a nitrate-responsive regulatory network in alfalfa and identifies MsHHO3 as a central regulator of symbiotic nitrogen fixation.

## Key findings

- MsHHO3 overexpression reduces nodule number and nitrogenase activity in alfalfa.
- MsHHO3 directly binds to the promoter of MsMYC2, a key gene in the JA signaling pathway.
- MsHHO3 regulates a transcriptional cascade involving hormone-related transcription factors and core nodulation genes.

## Abstract

Symbiotic nitrogen fixation (SNF), a unique nitrogen acquisition mechanism formed through the interaction between leguminous plants and rhizobia, plays a critical role in reducing dependence on chemical fertilizers. However, in alfalfa (Medicago sativa L.), the mechanisms underlying nitrate‐responsive SNF remain poorly understood. In this study, we elucidate a nitrate‐responsive regulatory network governing SNF in alfalfa and identify MsHHO3 as a key regulator. qRT‐PCR analysis revealed that MsHHO3 expression is significantly higher in the LAPIOSZELEI (LA) variety, which exhibits fewer nodules and lower nitrogenase activity, than in the TING SI (TS) variety, which shows a greater number of nodules and higher nitrogenase activity. Morphological characterization demonstrated that MsHHO3‐overexpressing lines exhibited significantly reduced nodule number, nodule fresh weight, and nitrogenase activity, whereas suppression of MsHHO3 by RNA interference (RNAi) in alfalfa resulted in an opposite phenotype. The CRISPR/Cas9 mutant of MtHHO3, a Medicago truncatula homolog of MsHHO3, displayed the similar phenotype as MsHHO3‐Ri. RNA‐seq, ChIP‐seq, and qRT‐PCR analysis showed that a set of nodulation‐associated genes were altered in MsHHO3‐overexpressing plants, as well as in MsHHO3‐Ri and mthho3 mutants. Among these genes, several hormone‐related TF‐encoding genes were directly regulated, including the JA signaling pathway master gene MsMYC2. EMSA and dual‐luciferase reporter assay further demonstrated that MsHHO3 can directly bind to the MsMYC2 promoter. We propose that MsHHO3 regulates SNF by modulating MsMYC2 and other intermediate TFs, orchestrating a transcriptional cascade that ensures precise fine‐tuning of the nodulation process. These findings provide novel mechanistic insights into nitrate‐responsive regulation of SNF in alfalfa.

HHO3 acts as a key regulator of nitrate‐responsive SNF by directly targeting several intermediate hormone‐related TF‐encoding genes (MsMYC2, Ms
RAV1, Ms
bHLH18, Ms
MYB108, Ms
MYB11) to initiate a transcriptional cascade, which in turn coordinates the expression of core nodulation genes YUC, PIN, and LBD for precise regulation.