| The Arabidopsis immutans (im) is variegated with green sectors and white sectors containing defective plastids due to a nuclear gene mutation. IM is a plastid terminal oxidase (PTOX) sharing similarity with mitochondrial alternative oxidase (AOX). In order to better understand IM's biological roles and variegation mechanism of im, I characterized im plants at different levels.; A structural model of IM, in which the diiron reaction center is composed of two conserved histidine and four glutamate residues, was tested by mutagenesis in vitro and in planta. It that these six conserved residues were found essential for IM activity and do not tolerate changes. Mutagenesis screening of 14 other conserved residues showed that two residues are essential, and four are important but not essential for IM activity. A 16 aa sequence uniquely present in PTOX was also found to be required for PTOX activity and stability. Overexpression of AOX in chloroplasts functionally rescues the variegation phenotype of im. And AOX deleted of the dimerization domain is more efficient to compensate im variegation phenotype, while addition of IM exon-8 sequence costs AOX function in chloroplasts.; A proteomic analysis suggested plastids in im white sectors were alive even without photosynthetic capacity. Defective plastids caused by photodamage induced a higher respiratory activity and various upregulated cytosolic proteins. Downregulated FtsZ1 level and microscopy analysis suggested that the division of photooxidized plastids were suppressed. Downregulation of IM to ∼3% of wild type levels did not compromise plant growth suggests that IM is normally in excess. Phenotypical observations indicated that IM does not significantly affect proplastids or differentiated plastids, but plays an essential role in early plastid differentiation stages.; A hypothesis was proposed to interpret im variegation mechanism. Proplastids develop into defective plastids due to photodamage without carotenoid protection; while some differentiating plastids escape photodamage and form chloroplasts due to shade effects. In a differentiating heteroplastidic cell, division of defective plastids was suppressed while chloroplasts maintain post-mitotic division. Consequently, homoplastidic and heteroplastidic differentiating cells with normal chloroplasts lead to green sector formation, while homoplastidic cells with defective plastids lead to white sector formation. |
