In-planta studies of magnesium chelatase

Tetrapyrrole biosynthesis is a key metabolic process in plants, not only because it leads to the production of heme, an essential co-factor for many enzymes, as well as chlorophylls, pigments necessary to the photosynthetic process, but also because this pathway has been implicated in retrograde signaling, a process by which the chloroplast exerts control over nuclear genes with products localized in the plastid. To date there has been extensive research in the characterization and activity of the enzymes involved in the tetrapyrrole biosynthesis, as well as regulation of, and flux through the pathway. -- In this field, studies utilizing plants generally employ seedlings and/or the use of herbicides such as norflurazon or lincomycin. However, interpreting the data from these studies can be ambiguous, because the use of herbicides and the process of seedling development both result in large pleiotropic effects. There has been relatively little work done to investigate magnesium chelatase in mature plants, after transition to autotrophic growth. The primary aim of the work presented in this thesis is to investigate the mechanism of magnesium chelatase activity in mature plants. This has been done in planta using Agrobacterium mediated transient over-expression of magnesium chelatase subunits CHLI, CHLD and CHLH. -- Over-expression of CHLD leads to severe chlorosis in Nicotiana benthamiana leaf tissue, comparable to that seen when a mutant of CHLI is over-expressed. Based on over-expression of CHLD deletion mutants, the chlorotic phenotype is attributable to the N-terminal half of the protein. CHLD induced chlorosis is almost completely rescued upon co-infiltration with CHLI, and interaction of the two subunits appears to involve a region of the C-terminal half of CHLD homologous to the Integrin I domain. Upon co-infiltration of CHLI with a CHLD construct mutagenized at the metal ion-dependent adhesion site (MIDAS), chlorosis is still observed.