Jie Ji

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.

Bacterial microcompartments (BMCs) are intracellular structures for compartmentalizing specific metabolic pathways in bacteria. As a unique type of BMCs, carboxysomes utilize protein shells to sequester ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase for efficient carbon dioxide (CO₂) fixation. This study aims to reconstruct an α-carboxysome in Saccharomyces cerevisiae and investigate its metabolic effects. Here, genes of the cso operon from Halothiobacillus neapolitanus, Calvin cycle-related enzyme phosphoribulokinase (PRK) from Spinacia oleracea, and two S. cerevisiae chaperone genes, HSP60 and HSP10, were introduced into S. cerevisiae. The engineered yeast strain demonstrated assembled and enzymatically active Rubisco, significant increase in ethanol production and reduction in the byproduct glycerol. Formation of the α-carboxysome structures was observed after purification by sucrose density gradient centrifugation. The engineered yeast strain harboring functional α-carboxysome has the potential for enhancing bioethanol production.

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 .