Journal: Biology

Article Title: Wheat Antipodal Cells with Polytene Chromosomes in the Embryo Sac Are Key to Understanding the Formation of Grain in Cereals

doi: 10.3390/biology11091340

Figure Lengend Snippet: Localization of cytochrome c in the cytoplasm of the wheat antipodal complex cells during PCD. A complex of antipodal cells ( a ). Cytochrome c in the mitochondria of a functioning antipodal cell ( b ) and in the cytoplasm of the antipodal cell during PCD ( c ). ant—antipodal, e—endosperm. Scale 20 µm.

Article Snippet: Rabbit polyclonal antibodies (1:100 dilution in PBS + 0.1% BSA) against cytochrome c (AS08 343A, Agrisera, Vännäs, Sweden) were used for the indirect immunocytochemical detection of mitochondria.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Identification of Physiological Substrates and Binding Partners of the Plant Mitochondrial Protease FTSH4 by the Trapping Approach

doi: 10.3390/ijms18112455

Figure Lengend Snippet: FTSH4 substrate trapping assay ( A ) Overview of FTSH4 substrate-trapping assay. Cartoon illustrating the experimental workflow; ( B ) Eluted fractions resolved on SDS-PAGE and stained with CBB. IMS—intermembrane space.

Article Snippet: Immunodecoration was made with antibodies against AtMic60 [ ], Slp1 [ ], Tim17-2 [ ], Tom40 [ ], FLAG-tag (Sigma-Aldrich, Steinheim, Germany, F1804), mtHsp70 (Agrisera, Vännäs, Sweden, AS08 347), cytochrome c (Agrisera, AS08 343A), FTSH4 (Agrisera, AS07 205).

Techniques: SDS Page, Staining

Journal: International Journal of Molecular Sciences

Article Title: Identification of Physiological Substrates and Binding Partners of the Plant Mitochondrial Protease FTSH4 by the Trapping Approach

doi: 10.3390/ijms18112455

Figure Lengend Snippet: Proteins co-purifying with FTSH4 TRAP.FLAG . Listed are all proteins that were specifically co-purifying with proteolytically inactive FTSH4 (proteins enriched at least two times in two independent biological replicates over the background samples ( Table S1 )). Functional categories and mitochondrial sub-localization were assigned based on the Uniprot database and the literature. AGI— Arabidopsis Gene ID.

Article Snippet: Immunodecoration was made with antibodies against AtMic60 [ ], Slp1 [ ], Tim17-2 [ ], Tom40 [ ], FLAG-tag (Sigma-Aldrich, Steinheim, Germany, F1804), mtHsp70 (Agrisera, Vännäs, Sweden, AS08 347), cytochrome c (Agrisera, AS08 343A), FTSH4 (Agrisera, AS07 205).

Techniques: Functional Assay

Journal: International Journal of Molecular Sciences

Article Title: Identification of Physiological Substrates and Binding Partners of the Plant Mitochondrial Protease FTSH4 by the Trapping Approach

doi: 10.3390/ijms18112455

Figure Lengend Snippet: Immunoblot analysis of FTSH4 substrate trapping assay samples. Mitochondria from control ( ftsh4-1 ) and ftsh4-1 FTSH4 TRAP.FLAG were solubilized with digitonin and subjected to immunoprecipitation with anti-FLAG affinity matrix. The precipitated proteins were immunoblotted with antibodies against the indicated proteins. IN—input (5%), FT—flow-through (5%), W—Wash, E—eluate (50%).

Article Snippet: Immunodecoration was made with antibodies against AtMic60 [ ], Slp1 [ ], Tim17-2 [ ], Tom40 [ ], FLAG-tag (Sigma-Aldrich, Steinheim, Germany, F1804), mtHsp70 (Agrisera, Vännäs, Sweden, AS08 347), cytochrome c (Agrisera, AS08 343A), FTSH4 (Agrisera, AS07 205).

Techniques: Western Blot, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: Identification of Physiological Substrates and Binding Partners of the Plant Mitochondrial Protease FTSH4 by the Trapping Approach

doi: 10.3390/ijms18112455

Figure Lengend Snippet: Novel proteolytic substrates of FTSH4 protease. ( A ) Degradation of Pam18-2 by FTSH4 following its in vitro import into mitochondria. Radiolabeled Pam18-2 was imported into mitochondria derived from either wild type or ftsh4-1 plants. The stability of newly imported precursor upon further incubation at 26 °C was analyzed by SDS-PAGE and autoradiography. Quantification of [ 35 S] Pam18-2 in mitochondria is represented in the lower panel. Newly imported Pam18-2 was set to 100%. Data represent mean ± SD of three independent experiments. * p < 0.02 and ** p < 0.003 ( t -student test); ( B ) Degradation of MPC4 processed form by FTSH4 following its in vitro import into mitochondria. Radiolabeled MPC4 was imported into mitochondria derived from either wild type or ftsh4-1 plants. The stability of newly imported MPC4 upon further incubation at 26 °C was analyzed by SDS-PAGE and autoradiography. Quantification of [ 35 S] MPC4 processed form in mitochondria is represented in the lower panel. Newly imported MPC4 was set to 100%. Data represent mean ± SD of three independent experiments. * p < 0.05 ( t -student test). m—MPC4 processed form, p—MPC4 precursor.

Article Snippet: Immunodecoration was made with antibodies against AtMic60 [ ], Slp1 [ ], Tim17-2 [ ], Tom40 [ ], FLAG-tag (Sigma-Aldrich, Steinheim, Germany, F1804), mtHsp70 (Agrisera, Vännäs, Sweden, AS08 347), cytochrome c (Agrisera, AS08 343A), FTSH4 (Agrisera, AS07 205).

Techniques: In Vitro, Derivative Assay, Incubation, SDS Page, Autoradiography

Journal: International Journal of Molecular Sciences

Article Title: Identification of Physiological Substrates and Binding Partners of the Plant Mitochondrial Protease FTSH4 by the Trapping Approach

doi: 10.3390/ijms18112455

Figure Lengend Snippet: FTSH4 degrades oxidatively damaged mitochondrial proteins. ( A ) Anti-DNP (dinitrophenyl hydrazone) immunoblot detection of carbonylated proteins co-precipitating with FTSH4 TRAP.FLAG . The cross-reaction of IgG light chain is marked with asterisk. ( B ) The degradation of mitochondrial carbonylated proteins by FTSH4 protease was assessed by immunoblot analysis of the levels of carbonylated proteins in mitochondrial extract from ftsh4-1 mutant incubated at 35 °C with or without FTSH4 protein. ( C ) Quantification of the remaining carbonylated proteins after 2 h incubation with or without FTSH4 protein, shown in ( B ). For each sample, the amount of carbonylated proteins was set to 100% at time point 0 h. Data represent mean ± SEM of three independent experiments. ** p < 0.03 ( t -student test).

Article Snippet: Immunodecoration was made with antibodies against AtMic60 [ ], Slp1 [ ], Tim17-2 [ ], Tom40 [ ], FLAG-tag (Sigma-Aldrich, Steinheim, Germany, F1804), mtHsp70 (Agrisera, Vännäs, Sweden, AS08 347), cytochrome c (Agrisera, AS08 343A), FTSH4 (Agrisera, AS07 205).

Techniques: Western Blot, Mutagenesis, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Conserved and Opposite Transcriptome Patterns during Germination in Hordeum vulgare and Arabidopsis thaliana

doi: 10.3390/ijms21197404

Figure Lengend Snippet: Mitochondrial dynamics during barley grain germination. ( A ) Oxygen consumption in whole embryos and isolated mitochondria. Oxygen consumption was examined in whole barley embryos and isolated mitochondria using a Clark-type oxygen electrode. Each data point represents an average of three to four independent measurements. ( B ) Changes in abundance of mitochondrial proteins. A representative image is shown from probing three independent isolated mitochondrial proteins using both 10 and 20 µg of isolated protein. The abundance of the protein is normalized to a value of 1 at 48 h and all other values are expressed in a relative manner. For NDUFS4 “a” and “b”, all values are relative to 12 h and 0 h, respectively. For B14.7 “a”, “b”, and “c”, all values are relative to 0 h, 0 h, and 24 h, respectively. The apparent molecular mass is indicated in kDa. NDUFS4 = 17 kDa subunit complex I, B14.7 = complex I subunit, SDH = subunit I of succinate dehydrogenase/complex II, RISP = Rieske FeS protein of complex III, COXII = cytochrome oxidase II of complex IV, ATPβ = β subunit of ATP synthase, Cyt c = cytochrome c, AOX = alternative oxidase, Tom20-3 = translocase of the outer membrane 20-3, Tom40 = translocase of the outer membrane 40, Porin = voltage-dependent anion channel, UCP = uncoupling protein, LETM1 = leucine zipper-EF-hand-containing transmembrane protein 1, Tim44 = translocase of the outer membrane 44. ( C ) The heatmap represents the abundance of 173 mitochondrial proteins at the indicated time points after seed inhibition. Quantification was performed by proteomic analysis (label-free quantification) of purified mitochondrial proteins, and abundances are represented by z-scored intensity-based absolute quantification (iBAQ) values. The position of several protein isoforms is indicated in the heatmap (see for details): CYC B/C, CYTOCHROM b/c subunit; ND, NADH DEHYDROGENASE; SDH, SUCCINATE DEHYDROGENASE; NDA/NDB, ALTERNATIVE NAD(P)H DEHYDROGENASE; COX, CYTOCHROME OXIDASE; LETM1, LEUCINE ZIPPER-EF-HAND-CONTAINING TRANSMEMBRANE PROTEIN 1.

Article Snippet: Antibodies were used against Porin [ ], AOX [ ], UCP [ ], LETM1 [ ], Tom20 [ ], RISP [ ], COXII (Agrisera, Vännäs, Sweden Cat #AS04 0543A), cytochrome c (Agrisera Vännäs, Sweden, Cat # AS08 343A), β-subunit of ATP synthase (Agrisera Vännäs, Sweden, Cat # AS05 085), and Tim44 [ ].

Techniques: Isolation, Membrane, Inhibition, Quantitative Proteomics, Purification