Accumulating evidence suggests that peptidoglycan, consistent with a bacterial cell wall, is synthesized around the chloroplasts of many photosynthetic eukaryotes, from glaucophyte algae to early-diverging land plants including pteridophyte ferns, but the biosynthetic pathway has not been demonstrated. Here, we employed mass spectrometry and enzymology in a two-fold approach to characterize the synthesis of peptidoglycan in chloroplasts of the moss Physcomitrium (Physcomitrella) patens. To drive the accumulation of peptidoglycan pathway intermediates, P.
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