Disruption of tobacco chloroplast clpP gene encoding a proteolytic subunit of ATP-dependent protease

Abstract

Clp is ATP-dependent protease, which is conserved in many organisms and is present in chloroplasts in higher plants. The protease consists of nuclear-encoded regulatory subunits and proteolytic subunits encoded both by nuclear and chloroplast genomes. We disrupted the chloroplast clpP gene (clpP1) using tobacco chloroplast transformation. Complete segregation of clpP1 was impossible, suggesting the essential function of clpP1 in cell survival. Although 50% of the wild-type gene was still remaining, transformants exhibited severe abnormality in leaf morphology. Chloroplasts consisted of two populations, wild-type-like chloroplasts, and small chloroplasts emitting high chlorophyll fluorescence. Thylakoid membranes of the small chloroplasts consisted exclusively of large stacks of tightly appressed membranes. Furthermore, the abnormal plastid structure was observed in several developmental stages, including etioplasts. These results suggest that Clp protease functions in multiple processes of chloroplast development, including a housekeeping function.