# Dissecting the Functional Interplay Between Heme Oxygenase LjHO1 and Leghemoglobins in Lotus japonicus Nodules

## TL;DR

This study explores how heme metabolism is regulated in legume nodules to support nitrogen fixation, focusing on the roles of leghemoglobins and heme oxygenase LjHO1.

## Contribution

The study reveals complementary roles of leghemoglobins and LjHO1 in heme homeostasis and symbiotic efficiency in legume nodules.

## Key findings

- Leghemoglobins are essential for heme accumulation and nitrogen fixation.
- LjHO1 fine-tunes heme turnover and contributes to nodule formation.
- Loss of LjHO1 slightly increases heme levels in nodules.

## Abstract

Legume root nodules require tightly regulated heme metabolism to support symbiotic nitrogen fixation (SNF). Leghemoglobins (Lbs) act as the primary heme reservoir, while heme oxygenase LjHO1 mediates heme degradation. In this study, we generated single and combinatorial mutants of Lbs and LjHO1 in Lotus japonicus using the CRISPR/Cas9 system. Our results show that Lbs are essential for heme synthesis and nitrogen fixation, whereas LjHO1 fine-tunes heme turnover and contributes to nodule formation. These findings highlight the complementary roles of Lbs and LjHO1 in maintaining nodule heme homeostasis and symbiotic efficiency.

Heme metabolism is crucial for the function and longevity of legume nodules, with leghemoglobins (Lbs) serving as the primary heme sink and heme oxygenase (HO) mediating heme degradation. However, the precise functional relationship between HO and Lbs remains unclear. Here, we show that Lotus japonicus HO1 (LjHO1) is strongly induced in early-stage Lb-deficient nodules, but its expression gradually decreases during nodule development. Subcellular localization analysis revealed that LjHO1 is plastid-localized in uninfected cells of lb123 mutant nodules, consistent with its localization in wild-type nodules. Using CRISPR/Cas9, we generated a quadruple ho1lb123 mutant lacking LjHO1 and all three Lb isoforms in L. japonicus. Phenotypic analyses revealed that Lbs deficiency predominantly impairs nitrogen fixation, whereas loss of LjHO1 further reduces nodule formation. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis revealed that loss of Lbs strongly decreased heme accumulation, whereas LjHO1 deficiency slightly increased heme levels in nodules. These results demonstrate that Lbs are essential for heme accumulation and nitrogen fixation, while LjHO1 fine-tunes heme turnover, highlighting their complementary roles in maintaining nodule heme homeostasis and symbiotic efficiency.