Heme is biosynthesized in legume root nodules to meet the demand for leghemoglobins (Lbs) and other heme-binding proteins. However, the main source of nodule heme remains unknown. Both the plant host and rhizobia possess a complete heme biosynthetic pathway, differing slightly in the production of 5-aminolevulinic acid (ALA), a key regulatory step catalyzed by glutamyl-tRNA reductase (GluTR) in the plant and by HemA in the rhizobia. Transcriptomic analysis revealed that many plant heme biosynthetic genes, including GluTR2 but not GluTR1, are upregulated in nodules compared to roots, whereas expression of related rhizobial genes, including both HemA1 and HemA2, is generally inhibited under symbiotic conditions compared to free-living conditions. Knockout of Lotus japonicus GluTR2, but not of HemA1 and HemA2, led to a significant decrease (∼50%) in nodule heme content. The stable heterozygous mutant of GluTR1 or transient knockdown of GluTR1 exhibited a ∼20% reduction in nodule heme content. Overexpression of Fluorescent in blue light (FLU), a feedback inhibitor of GluTR activity, caused a much greater reduction in nodule heme content (∼75%) and an increased level of apo-Lb and, in combination with the hemA1 hemA2 mutant, a drastic inhibition of nitrogenase activity (>90%). This study provides genetic evidence supporting a major role of plant GluTRs in coordinating heme biosynthesis between the two symbionts by supplying heme to assemble with cytoplasmic apo-Lbs and by providing ALA for heme synthesis in the bacteroids.

Plant lipid transfer proteins (LTPs), belonging to the pathogenesis-related protein 14 family, participate in plant immune response to biotic stress. LTP1 from Vigna unguiculata was previously shown to be able to suppress infection by cowpea mosaic virus and soybean mosaic virus. However, whether cowpea LTP1 participates in the plant resistance to other plant pathogens remains unclear. The present study analyzed the role of LTP1 in plant resistance to eukaryotic pathogens. We observed that LTP1 overexpression in cowpea and tobacco significantly reduced the lesion areas and biomass of the fungus Botrytis cinerea and oomycete Phytophthora capsici. A protein lipid overlay assay showed that LTP1 bound phosphatidic acid and phosphatidylinositol (4,5)-bisphosphate, but LTP1 ^3A , with three amino acids in the lipid-binding domain being mutated to alanine, lost its lipid-binding ability. Consistently, overexpression of LTP1 ^3A did not influence lesion area and pathogen biomass in cowpea and tobacco plants after inoculation with B. cinerea at 48 h postinoculation. LTP1 heterologous expression in tobacco induced a significant increase in intracellular calcium, inositol 1,4,5-trisphosphate (IP ₃ ) levels, and abscisic acid contents, leading to more significant stomatal closure after B. cinerea infection. Overall, our findings suggest that cowpea LTP1 participates in the plant defense response through interacting with specific phospholipids, thereby interfering with pathological processes such as IP ₃ -mediated calcium signaling and stomatal movement. [ ] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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.

Heme is essential for symbiotic nitrogen fixation (SNF), but the contribution of ferrochelatase (FC)-mediated heme biosynthesis to nodule function remains poorly understood. Here, we characterized heme metabolism and the FC gene family in Lotus japonicus nodules. Heme and its precursor protoporphyrin IX (PPIX) accumulated preferentially in mature nodules and declined during nodule senescence and in the leghemoglobin-deficient lb123 mutant. Four FC genes, LjFC1–LjFC4, were identified, and promoter-GUS analysis revealed distinct spatial patterns among the four paralogs. LjFC1–LjFC3 promoter activities were mainly associated with vascular and cortical tissues or uninfected cells, whereas LjFC4 promoter activity was detected in both infected and uninfected nodule cells. All four LjFC proteins localized to plastids in root nodules. Moreover, pharmacological inhibition of FC activity with N-PPIX reduced nodule heme levels and impaired nodule development. Collectively, these findings establish a plastid-associated localization of the LjFC family in root nodules and support an association between FC-related heme metabolism and nodule development.

Heme is essential for symbiotic nitrogen fixation (SNF), but the contribution of ferrochelatase (FC)-mediated heme biosynthesis to nodule function remains poorly understood. Here, we characterized heme metabolism and the FC gene family in Lotus japonicus nodules. Heme and its precursor protoporphyrin IX (PPIX) accumulated preferentially in mature nodules and declined during nodule senescence and in the leghemoglobin-deficient lb123 mutant. Four FC genes, LjFC1–LjFC4, were identified, and promoter-GUS analysis revealed distinct spatial patterns among the four paralogs. LjFC1–LjFC3 promoter activities were mainly associated with vascular and cortical tissues or uninfected cells, whereas LjFC4 promoter activity was detected in both infected and uninfected nodule cells. All four LjFC proteins localized to plastids in root nodules. Moreover, pharmacological inhibition of FC activity with N-PPIX reduced nodule heme levels and impaired nodule development. Collectively, these findings establish a plastid-associated localization of the LjFC family in root nodules and support an association between FC-related heme metabolism and nodule development.