Effects of Tobacco Plastid Division Genes NtFtsZ1 and NtFtsZ2 on the Division and Morphology of Chloroplasts

As an important group of plant cellular organelles, the molecular mechanism of plastid division is poorly understood. Recent studies have revealed that the homologs of ftsZ gene, an essential prokaryotic cell division gene, are involved in plastid division process of plant cells. Antisense and sense expression constructions were employed to investigate the functions of the two ftsZ genes, NtFtsZ1 and NtFtsZ2, in transgenic Nicotiana tabacum L. plants. Although antisense expression of,NtFtsZs reduced the native protein level obviously, the size and number of chloroplasts in transgenic tobacco plants had no effect. In contrast, overexpression of NtFtsZs in transgenic plants strikingly changed the number and morphology of chloroplasts. Even only 1 - 2 huge chloroplasts could be seen in the mesophyll cells of some overexpression transgenic plants. Analyses of chloroplast ultrastructures and chlorophyll content of different transgenic plants suggested that NtFtsZs gene have no direct influence on the normal development and function of chloroplasts. ne changes in chloroplast morphology must be a compensation for the change in chloroplast number. The different phenotypes of chloroplasts in antisense and sense transgenic plants implied that different members from the same ftsZ gene family may have similar function in controlling plastid division. Meanwhile, the changes of chloroplast morphology in sense transgenic plants represented the possible plastoskeleton function of ftsZ in higher plant.

Localization of Two GFP_tagged Tobacco Plastid Division Protein NtFtsZs in Escherichia coli

Two plastid division genes, NtFtsZ1 and NtFtsZ2 isolated from Nicotiana tabacum L. were fused with gfp and expressed in Escherichia coli . The regular localizations of full length NtFtsZs∶GFP along the filamentous bacteria indicated that the NtFtsZs could recognize the potential division sites in E. coli and be polymerized with heterogeneous FtsZ from bacteria. The overexpression of NtFtsZs ∶ gfp inhibited the division of host strain cells and resulted in the long filamentous bacterial morphology. These results suggested that eukaryotic ftsZs have similar function to their prokaryotic homologs. Meanwhile, the different deletions of motifs of NtFtsZs are also employed to investigate the functions of these proteins in E. coli . The results showed that the C_terminal domains of NtFtsZs were related to the correct localization of NtFtsZs in E. coli and the N_terminal domains of NtFtsZs were responsible for the polymerization of homogeneous and heterogeneous FtsZ proteins. The significance of these results in understanding the functions of NtFtsZs in plastid division were discussed.

Arabidopsis FtsZ2-1 and FtsZ2-2 Are Functionally Redundant, But FtsZ-Based Plastid Division Is Not Essential for Chloroplast Partitioning or Plant Growth and Development

FtsZ1 and FtsZ2 are phylogenetically distinct families of FtsZ in plants that co-localize to mid-plastid rings and facilitate division of chloroplasts. In plants, altered levels of either FtsZ1 or FtsZ2 cause dose-dependent defects in chloroplast division; thus, studies on the functional relationship between FtsZgenes require careful manipulation of FtsZ levels in vivo. To define the functional relationship between the two FtsZ2 genes in Arabidopsis thaliana, FtsZ2-1 and FtsZ2-2, we expressed FtsZ2-1 in an ftsZ2-2 null mutant, and vice versa, and determined whether the chloroplast division defects were rescued in plants expressing different total levels of FtsZ2. Full rescue was observed when either the FtsZ2-1 or FtsZ2-2 level approximated total FtsZ2 levels in wild-type （WT）. Additionally, FtsZ2-2 interacts with ARC6, as shown previously for FtsZ2- 1. These data indicate that FtsZ2-1 and FtsZ2-2 are functionally redundant for chloroplast division in Arabidopsis. To rigorously validate the requirement of each FtsZ family for chloroplast division, we replaced FtsZ1 with FtsZ2 in vivo, and vice versa, while maintaining the FtsZ level in the transgenic plants equal to that of the total level in WT. Chloroplast division defects were not rescued, demonstrating conclusively that FtsZ1 and FtsZ2 are non-redundant for maintenance of WT chloroplast numbers. Finally, we generated ftsZtriple null mutants and show that plants completely devoid of FtsZ protein are viable and fertile. As plastids are presumably essential organelles, these findings suggest that an FtsZ-independent mode of plastid partitioning may occur in higher plants.

OR^His, a Natural Variant of OR, Specifically Interacts with Plastid Division Factor ARC3 to Regulate Chromoplast Number and Carotenoid Accumulation

Chromoplasts are colored plastids that synthesize and store massive amounts of carotenoids.Chromoplast number and size define the sink strength for carotenoid accumulation in plants.However,nothing is known about the mechanisms controlling chromoplast number.Previously,a natural allele of Orange(OR),OR^His,was found to promote carotenoid accumulation by activating chromoplast differentiation and increasing carotenoid biosynthesis,but cells in orange tissues in melon fruit and cauliflower OR mutant have only one or two enlarged chromoplasts.In this study,we investigated an OR^His variant of Arabidopsis OR,genetically mimicking the melon OR^His allele,and found that it also constrains chromoplast number in Arabidopsis calli.Both in vitro and in vivo experiments demonstrate that OR^His specifically interacts with the Membrane Occupation and Recognition Nexus domain of ACCUMULATION AND REPLICATION OF CHLOROPLASTS 3(ARC3),a crucial regulator of chloroplast division.We further showed that OR^His interferes with the interaction between ARC3 and PARALOG OF ARC6(PARC6),another key regulator of chloroplast division,suggesting a role of OR^His in competing with PARC6 for binding to ARC3 to restrict chromoplast number.Overexpression or knockout of ARC3 in Arabidopsis OR^His plants significantly alters total carotenoid levels.Moreover,overexpression of the plastid division factor PLASTID DIVISION 1 greatly enhances carotenoid accumulation.These division factors likely alter carotenoid levels via their influence on chromoplast number and/or size.Taken together,our findings provide novel mechanistic insights into the machinery controlling chromoplast number and highlight a potential new strategy for enhancing carotenoid accumulation and nutritional value in food crops.

A Nucleus-encoded Topological Specificity Factor PpMinE in Physcomitrella patens has Conserved Function Similar to Its Chloroplast-encoded Ancestor

A nucleus-encoded MinE gene, designated PpMinE, from Physcomitrella patens was identified using RT-PCR. The presence of both N- and C-terminal extensions in PpMinE protein suggested its cyanobacterial origin. The transient expression of PpMinE using green fluorescent protein fusion in tobacco （Nicotiana tabacum L.） indicated that the PpMinE was a chloroplast-targeted protein. Overexpression of PpMinE in Escherichia coli caused division site misplacement and minicell formation, suggesting evolutionary functional conservation of MinE during plant phylogenesis. According to the phylogenetic tree, PpMinE protein has a close relationship with the highland plants, which suggests that the transfer events of MinE gene from plastid to nucleus might have occurred before the origin of the land plants.