Symbiosomal Proteomic Analysis Reveals the Implication of Endosomal Regulators and CAPs in the Formation of Peanut Nodules.

Arachis hypogaea (peanut) is an important leguminous crop that obtains nitrogen through symbiotic nitrogen fixation with rhizobia, with root nodules serving as the site of this symbiosis. Although the cytological characteristics and ultrastructure of root nodules in model leguminous plants have been well elucidated, research progress on peanut root nodules remains relatively limited. In this study, we characterized the spatiotemporal developmental pattern of peanut root nodules through microscopic imaging and ultrastructural analysis. Furthermore, we isolated symbiosome-enriched fractions from peanut nodules for proteomic analysis and identified 340 and 182 peanut proteins in a comprehensive proteome atlas of the peanut symbiosome membrane (SM) and peribacteroid space (PBS), respectively. Notably, our analysis revealed a significant enrichment of endosomal regulators in the SM and CAP family proteins (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins) in the PBS. Finally, we demonstrated that AhCAP21 specifically localizes to the symbiosome, and the SM-localized AhRabA2a is essential for proper symbiosome development. Together, these findings advance our understanding of peanut nodule development and provide insights into the protein compositions and regulators in symbiosome biogenesis in peanut nodules.