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CTP synthases (CTPS) comprise a protein family of the five members CTPS1-CTPS5 in Arabidopsis, all located in the cytosol. Specifically, downregulation of CTPS2 by amiRNA technology results in plants with defects in chlorophyll accumulation and photosynthetic performance early in development. CTP and its deoxy form dCTP are present at low levels in developing seedlings. Thus, under conditions of fast proliferation, the synthesis of CTP (dCTP) can become a limiting factor for RNA and DNA synthesis. The higher sensitivity of ami-CTPS2 lines toward the DNA-Gyrase inhibitor ciprofloxacin, together with reduced plastid DNA copy number and 16S and 23S chloroplast ribosomal RNA support this view. High expression and proposed beneficial biochemical features render CTPS2 the most important isoform for early seedling development. In addition, CTPS2 was identified as an essential enzyme in embryo development before, as knock-out mutants were embryo lethal. In line with this, ami-CTPS2 lines also exhibited reduced seed numbers per plant.

Thylakoids are a formation of flattened membrane vesicles and protein complexes found in cyanobacteria, algae and plants. In the chloroplasts of land plants the thylakoid membrane systems form a network of densely packed stacks called grana lamellae, which are connected by unstacked stroma lamellae. Photosystem II is mainly localized in the appressed grana region, while photosystem I and the ATP synthase complexes are enriched in the stroma lamellae. The cytochrome b6/f complex is distributed laterally throughout both stacked and unstacked membrane regions. The photosynthetic complexes consist of integral and peripheral proteins. The first part of this protocol (A) shows how to fractionate thylakoids into grana and stroma lamellae. The second part of this protocol (B) shows how to distinguish between strong hydrophobic integral membrane associations and weak electrostatic membrane and/or membrane complex associations. As it is necessary to specifically detect the protein of interest in the fractions, a specific antibody raised against the protein of interest or a complemented null mutant of a structural component expressing a tagged fusion protein would be of great advantage. The last part of this protocol (C) shows, how to investigate the topology of integral and peripheral proteins. This method requires a specific antibody for the protein of interest. For integral membrane proteins peptide-specific antibodies or epitope-tagged versions are required. The protocol is suitable for the investigation of low molecular weight proteins (LMW) below 5 kDa (Torabi et al., 2014).

Oxygen-evolving chloroplasts possess their own iron-sulfur cluster assembly proteins including members of the SUF (sulfur mobilization) and the NFU family. Recently, the chloroplast protein HCF101 (high chlorophyll fluorescence 101) has been shown to be essential for the accumulation of the membrane complex Photosystem I and the soluble ferredoxin-thioredoxin reductases, both containing [4Fe-4S] clusters. The protein belongs to the FSC-NTPase ([4Fe-4S]-cluster-containing P-loop NTPase) superfamily, several members of which play a crucial role in Fe/S cluster biosynthesis. Although the C-terminal ISC-binding site, conserved in other members of the FSC-NTPase family, is not present in chloroplast HCF101 homologues using Mössbauer and EPR spectroscopy, we provide evidence that HCF101 binds a [4Fe-4S] cluster. 55Fe incorporation studies of mitochondrially targeted HCF101 in Saccharomyces cerevisiae confirmed the assembly of an Fe/S cluster in HCF101 in an Nfs1-dependent manner. Site-directed mutagenesis identified three HCF101-specific cysteine residues required for assembly and/or stability of the cluster. We further demonstrate that the reconstituted cluster is transiently bound and can be transferred from HCF101 to a [4Fe-4S] apoprotein. Together, our findings suggest that HCF101 may serve as a chloroplast scaffold protein that specifically assembles [4Fe-4S] clusters and transfers them to the chloroplast membrane and soluble target proteins. [ABSTRACT FROM AUTHOR]

Like bacteria, plastids contain sigma factors for promoter binding and transcription initiation. Accumulating evidence suggests that members of the plant sigma factor family can have specialized non-redundant roles in terms of promoter preference in various developmental and environmental situations. To specify regulatory determinants, we have chosen pairwise exchange of portions of Arabidopsis sigma coding regions, followed by transformation of the chimeric constructs into a sigma 6 knockout line. The resulting phenotypes and plastid RNA patterns point to an important though not exclusive role for the highly variable N-terminal portion of plant sigma proteins. [ABSTRACT FROM AUTHOR]

The flu mutant of Arabidopsis thaliana overaccumulates in the dark the immediate precursor of chlorophyllide, protochlorophyllide (Pchlide), a potent photosensitizer, that upon illumination generates singlet oxygen (1O2). Once 1O2 has been released in plastids of the flu mutant, mature plants stop growing, while seedlings die. Several suppressor mutations, dubbed singlet oxygen-linked death activator (soldat), were identified that specifically abrogate 1O2-mediated stress responses in young flu seedlings without grossly affecting 1O2-mediated stress responses of mature flu plants. One of the soldat mutations, soldat8, was shown to impair a gene encoding the SIGMA6 factor of the plastid RNA polymerase. Reintroduction of a wild-type copy of the SOLDAT8 gene into the soldat8/flu mutant restored the phenotype of the flu parental line. In contrast to flu, seedlings of soldat8/flu did not bleach when grown under non-permissive dark/light conditions, despite their continuous overaccumulation of the photosensitizer Pchlide in the dark. The activity of SIGMA6 is confined primarily to the very early stage of seedling development. Inactivation of SIGMA6 in soldat8 mutants disturbed plastid homeostasis, drastically reduced the non-photochemical quenching capacity and enhanced the light sensitivity of young soldat8 seedlings. Surprisingly, after being grown under very low light, soldat8 seedlings showed an enhanced resistance against a subsequent severe light stress that was significantly higher than in wild-type seedlings. In order to reach a similar enhanced stress resistance, wild-type seedlings had to be exposed to a brief higher light treatment that triggered an acclimatory response. Such a mild pre-stress treatment did not further enhance the stress resistance of soldat8 seedlings. Suppression of 1O2-mediated cell death in young flu/soldat8 seedlings seems to be due to a transiently enhanced acclimation at the beginning of seedling development caused by the initial disturbance of plastid homeostasis. [ABSTRACT FROM AUTHOR]