Dataset Information

The Formation of Stromules In Vitro from Chloroplasts Isolated from Nicotiana benthamiana.

ABSTRACT: Stromules are stroma-containing tubules that have been observed to emanate from the main plastidic body in vivo. These structures have been shown to require cytoskeletal components for movement. Though numerous studies have shown a close association with the endoplasmic reticulum, nucleus, mitochondria, and other plastids, the mechanism of formation and their overall function remain unknown. A limiting factor in studying these structures has been the lack of a reconstituted system for in vitro stromule formation. In this study, stromule formation was induced in vitro by adding a plant extract fraction that is greater than 100 kDa to a population of isolated chloroplasts. Kinetic measurements show that stromule formation occurs within ~10 seconds after the addition of the plant extract fraction. Heat inactivation and apyrase treatment reveal that the stromule stimulating compound found in the extract fraction is a protein or protein complex 100 kDa or greater. The formation of the stromules in vitro with isolated chloroplasts and a concentrated fraction of cell extract opens an avenue for the biochemical dissection of this process that has heretofore been studied only in vivo.

SUBMITTER: Ho J

PROVIDER: S-EPMC4739594 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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The Formation of Stromules In Vitro from Chloroplasts Isolated from Nicotiana benthamiana.

Ho Jonathan J Theg Steven M SM

PloS one 20160203 2

Stromules are stroma-containing tubules that have been observed to emanate from the main plastidic body in vivo. These structures have been shown to require cytoskeletal components for movement. Though numerous studies have shown a close association with the endoplasmic reticulum, nucleus, mitochondria, and other plastids, the mechanism of formation and their overall function remain unknown. A limiting factor in studying these structures has been the lack of a reconstituted system for in vitro s ...[more]

PMID: 26840974

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Project description:Engineering plants to synthesize nitrogenase and assimilate atmospheric N2 will reduce crop dependency on industrial N fertilizers. This technology can be achieved by expressing prokaryotic nitrogen fixation gene products for the assembly of a functional nitrogenase in plants. NifB is a critical nitrogenase component since it catalyzes the first committed step in the biosynthesis of all types of nitrogenase active-site cofactors. Here, we used a library of 30 distinct nifB sequences originating from different phyla and ecological niches to restore diazotrophic growth of an Azotobacter vinelandii nifB mutant. Twenty of these variants rescued the nifB mutant phenotype despite their phylogenetic distance to A. vinelandii. Because multiple protein interactions are required in the iron-molybdenum cofactor (FeMo-co) biosynthetic pathway, the maturation of nitrogenase in a heterologous host can be divided in independent modules containing interacting proteins that function together to produce a specific intermediate. Therefore, nifB functional modules composed of a nifB variant, together with the A. vinelandii NifS and NifU proteins (for biosynthesis of NifB [Fe4S4] clusters) and the FdxN ferredoxin (for NifB function), were expressed in Nicotiana benthamiana chloroplasts and mitochondria. Three archaeal NifB proteins accumulated at high levels in soluble fractions of chloroplasts (Methanosarcina acetivorans and Methanocaldococcus infernus) or mitochondria (M. infernus and Methanothermobacter thermautotrophicus). These NifB proteins were shown to accept [Fe4S4] clusters from NifU and were functional in FeMo-co synthesis in vitro. The accumulation of significant levels of soluble and functional NifB proteins in chloroplasts and mitochondria is critical to engineering biological nitrogen fixation in plants. IMPORTANCE Biological nitrogen fixation is the conversion of inert atmospheric dinitrogen gas into nitrogen-reactive ammonia, a reaction catalyzed by the nitrogenase enzyme of diazotrophic bacteria and archaea. Because plants cannot fix their own nitrogen, introducing functional nitrogenase in cereals and other crop plants would reduce our strong dependency on N fertilizers. NifB is required for the biosynthesis of the active site cofactors of all nitrogenases, which arguably makes it the most important protein in global nitrogen fixation. NifB functionality is therefore a requisite to engineer a plant nitrogenase. The expression of nifB genes from a wide range of prokaryotes into the model diazotroph Azotobacter vinelandii shows a surprising level of genetic complementation suggestive of plasticity in the nitrogenase biosynthetic pathway. In addition, we obtained NifB proteins from both mitochondria and chloroplasts of tobacco that are functional in vitro after reconstitution by providing [Fe4S4] clusters from NifU, paving the way to nitrogenase cofactor biosynthesis in plants.

Dataset Information

The Formation of Stromules In Vitro from Chloroplasts Isolated from Nicotiana benthamiana.

Publications

The Formation of Stromules In Vitro from Chloroplasts Isolated from Nicotiana benthamiana.

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