In vivo quantitative relationship between plastid division proteins FtsZ1 and FtsZ2 and identification of ARC6 and ARC3 in a native FtsZ complex.

FtsZ1 and FtsZ2 are phylogenetically distinct homologues of the tubulin-like bacterial cell division protein FtsZ that play major roles in the initiation and progression of plastid division in plant cells. Both proteins are components of a mid-plastid ring, the Z-ring, which functions as a contractile ring on the stromal surface of the chloroplast IEM (inner envelope membrane). FtsZ1 and FtsZ2 have been shown to interact, but their in vivo biochemical properties are largely unknown. To gain insight into the in vivo biochemical relationship between FtsZ1 and FtsZ2, in the present study we investigated their molecular levels in wild-type Arabidopsis thaliana plants and endogenous interactions in Arabidopsis and pea. Quantitative immunoblotting and morphometric analysis showed that the average total FtsZ concentration in chloroplasts of 3-week-old Arabidopsis plants is comparable with that in Escherichia coli. FtsZ levels declined as plants matured, but the molar ratio between FtsZ1 and FtsZ2 remained constant at approx. 1:2, suggesting that this stoichiometry is regulated and functionally important. Density-gradient centrifugation, native gel electrophoresis, gel filtration and co-immunoprecipitation experiments showed that a portion of the FtsZ1 and FtsZ2 in Arabidopsis and pea chloroplasts is stably associated in a complex of approximately 200-245 kDa. This complex also contains the FtsZ2-interacting protein ARC6 (accumulation and replicatioin of chloroplasts 6), an IEM protein, and analysis of density-gradient fractions suggests the presence of the FtsZ1-interacting protein ARC3. Based on the mid-plastid localization of ARC6 and ARC3 and their postulated roles in promoting and inhibiting chloroplast FtsZ polymer formation respectively, we hypothesize that the FtsZ1-FtsZ2-ARC3-ARC6 complex represents an unpolymerized IEM-associated pool of FtsZ that contributes to the dynamic regulation of Z-ring assembly and remodelling at the plastid division site in vivo.

Proteomic study of the Arabidopsis thaliana chloroplastic envelope membrane utilizing alternatives to traditional two-dimensional electrophoresis.

With the completion of the sequencing of the Arabidopsis genome and with the significant increase in the amount of other plant genome and expressed sequence tags (ESTs) data, plant proteomics is rapidly becoming a very active field. We have pursued a high-throughput mass spectrometry-based proteomics approach to identify and characterize membrane proteins localized to the Arabidopsis thaliana chloroplastic envelope membrane. In this study, chloroplasts were prepared from plate- or soil-grown Arabidopsis plants using a novel isolation procedure, and "mixed" envelopes were subsequently isolated using sucrose step gradients. We applied two alternative methodologies, off-line multidimensional protein identification technology (Off-line MUDPIT) and one-dimensional (1D) gel electrophoresis followed by proteolytic digestion and liquid chromatography coupled with tandem mass spectrometry (Gel-C-MS/MS), to identify envelope membrane proteins. This proteomic study enabled us to identify 392 nonredundant proteins.

THE PLASTID DIVISION MACHINE.

Plastid division is essential for the maintenance of plastid populations in cells undergoing division and for the accumulation of large chloroplast numbers in photosynthetic tissues. Although the mechanisms mediating plastid division are poorly understood, ultrastructural studies imply this process is accomplished by a dynamic macromolecular machine organized into ring structures at the plastid midpoint. A key component of the engine that powers this machine is the motor-like protein FtsZ, a cytoskeletal GTPase of endosymbiotic origin that forms a ring at the plastid division site, similar to the function of its prokaryotic relatives in bacterial cytokinesis. This review considers the phylogenetic distribution and structural properties of two recently identified plant FtsZ protein families in the context of their distinct roles in plastid division and describes current evidence regarding factors that govern their placement at the division site. Because of their evolutionary and mechanistic relationship, the process of bacterial cell division provides a valuable, though incomplete, paradigm for understanding plastid division in plants.