CRUMPLED LEAF (CRL) homologs of Physcomitrella patens are involved in the complete separation of dividing plastids.

Plastid division is controlled by numerous nuclear genes. Arabidopsis thaliana CRUMPLED LEAF (AtCRL) is a plastid division-related gene, and the crl mutant exhibits a dwarf phenotype with abnormal cell division and a significant reduction in plastid numbers. However, the function of AtCRL is not fully understood. Here, we identified and characterized two AtCRL homologs, PpCRL1 and PpCRL2, in the moss Physcomitrella patens. PpCRL1 and PpCRL2 shared 77% amino acid identity with each other and 47% identity with AtCRL. Single PpCRL1 or -2 gene knockout (KO) mutants could not be distinguished from the wild-type mosses, but PpCRL1 and -2 double KO mutants displayed growth retardation of protonemata and gametophores and harbored approximately 10 large chloroplasts per cell. This indicates that PpCRL1 and PpCRL2 have redundant functions in chloroplast division and plant growth. Unlike the A. thaliana crl mutants, however, the PpCRL double KO mutants did not display abnormal orientation of the cell division plane. Complementation experiments showed that AtCRL partially rescued the defects in chloroplast size and number of the PpCRL double KO mutant. This suggests that PpCRL has a similar, but not identical, function to AtCRL. Time-lapse microscopic observation of the double PpCRL KO mutants revealed that some dumbbell-shaped chloroplasts failed to complete division at the late stage of plastid division; enlarged chloroplasts were thus generated. This strongly suggests that PpCRLs are involved in the complete separation of dividing chloroplasts.