It is believed that the plastids in green plants lost peptidoglycan (i.e., a bacterial cell wall-containing D-amino acids) during their evolution from an endosymbiotic cyanobacterium. Although wall-like structures could not be detected in the plastids of green plants, the moss Physcomitrella patens has the genes required to generate peptidoglycan (Mur genes), and knocking out these genes causes defects in chloroplast division. Here, we generated P. patens knockout lines (ΔPp-ddl) for a homolog of the bacterial peptidoglycan-synthetic gene encoding D-Ala:D-Ala ligase. ΔPp-ddl had a macrochloroplast phenotype, similar to other Mur knockout lines. The addition of D-Ala-D-Ala (DA-DA) to the medium suppressed the appearance of giant chloroplasts in ΔPp-ddl, but the addition of L-Ala-L-Ala (LA-LA), DA-LA, LA-DA, or D-Ala did not. Recently, a metabolic method for labeling bacterial peptidoglycan was established using ethynyl-DA-DA (EDA-DA) and click chemistry to attach an azidemodified fluorophore to the ethynyl group. The ΔPp-ddl line complemented with EDA-DA showed that moss chloroplasts are completely surrounded by peptidoglycan. Our findings strongly suggest that the moss plastids have a peptidoglycan wall containing D-amino acids. By contrast, no plastid phenotypes were observed in the T-DNA tagged ddl mutant lines of Arabidopsis thaliana.

In the moss Physcomitrella patens, 10 Mur genes involved in peptidoglycan biosynthesis were found, and the MurE and Pbp genes are related to plastid division. Although the MraY and MurG genes were missing in our previous expressed sequence tag screening, they were discovered in the P. patens genome in this study, indicating that P. patens has a full set of genes capable of synthesizing peptidoglycan. In addition, a second MurA gene (PpMurA2) was found. Whereas Northern analyses indicated that PpMurA1, PpMurG and PpMraY were expressed, transcripts of PpMurA2 were detected only when RT-PCR was employed. Whereas GFP fusion proteins with either PpMurA1 or PpMraY were detected in chloroplasts, the PpMurA2 fusion proteins were located in the cytoplasm. Protonema cells in the wild-type plants had an average of 46 chloroplasts. PpMurA1 gene-disrupted lines had <10 chloroplasts, whereas approximately 30 chloroplasts existed in the PpMurA2 knockout lines. The PpMurA1/A2 double-knockout lines had only a few macrochloroplasts, suggesting a redundant function for these two genes. Disruption of the PpMraY gene in P. patens resulted in the appearance of macrochloroplasts. Anabaena MraY fused to the N-terminal region of PpMraY and A. thaliana MraY could complement the macrochloroplast phenotype in the PpMraY knockout line. Electron microscopic observations showed no obvious differences in the shape or stacking of thylakoid membranes between all knockout transformants and wild-type plants, suggesting that these Mur genes are related only to plastid division in moss.