A nodule-specific plant cysteine proteinase, AsNODF32, is involved in nodule senescence and nitrogen fixation activity of the green manure legume Astragalus sinicus

Asnodf32, encoding a nodule-specific cysteine proteinase in Astragalus sinicus, is probably involved in nodule senescence. To obtain direct evidence of its role in nodule senescence, Agrobacterium rhizogenes-mediated RNA interference was applied to A. sinicus hairy roots. Real-time qRT-PCR was used to estimate the efficiency of suppression. The senescent phenotype of transgenic nodules was examined with paraffin-embedded slides, TUNEL (TdT-mediated dUTP nick-end labeling) assay, and transmission electron microscopy, and the bacteroid nitrogen fixation activity was also measured. It was found that silencing of Asnodf32 delayed root nodule and bacteroid senescence. The period of bacteroid active nitrogen fixation was significantly extended. Interestingly, nodules enlarged in length were also observed on Asnodf32-silenced hairy roots. The results reported here indicate that Asnodf32 plays an important role in the regulation of root nodule senescence.