http://biol.unt.edu/~rmittler/Papers/Reactive%20oxygen%20gene%20network%20of%20plants.pdf

Reactive oxygen gene network of plants

Blue native PAGE (BN-PAGE) can be used for one-step isolation of protein complexes from biological membranes and total cell and tissue homogenates. It can also be used to determine native protein masses and oligomeric states and to identify physiological protein–protein interactions. Native complexes are recovered from gels by electroelution or diffusion and are used for 2D crystallization and electron microscopy or analyzed by in-gel activity assays or by native electroblotting and immunodetection. In this protocol, we describe methodology to perform BN-PAGE followed by (i) native extraction or native electroblotting of separated proteins, or (ii) a second dimension of tricine-SDS-PAGE or modified BN-PAGE, or (iii) a second dimension of isoelectric focusing (IEF) followed by a third dimension of tricine-SDS-PAGE for the separation of subunits of complexes. These protocols for 2D and 3D PAGE can be completed in 2 and 3 days.

Blue native PAGE

In addition to the often-cited advantages of using Arabidopsis thaliana as a model system in plant biological research (1), Arabidopsis has many additional characteristics that make it an attractive experimental organism for studying lea d (Pb) accumulation and tolerance in plants. These include its fortuitous familial relationship to many known metal hyperaccumulators (Brassicaceae), as well as similar Pbaccumulation patterns to most other plants. Using nutrient-agar plates, hydroponic culture, and Pb-contaminated soils as growth media, we found significant variation in Arabidopsis thaliana ecotypes in accumulation and tolerance of Pb. In addition, we have found that Pb accumulation is not obligatorily linked with Pb tolerance, suggesti ng that different genetic factors control these two processes. We also screened ethyl methanesulfonate-mutagenized M2 populations and identified several Pb-accumulating mutants. Current characterization of these mutants indicates that their phenotypes are likely due to alteration of general metal ion uptake or translocation processes since these mutants also accumulate many other metals in shoots. We expect that further characterization of the ecotypes and mutants will shed light on the basic genetic and physiological underpinnings of plant-based Pb remediation. 7. Aromatic nitroreduction of acifluorfen in soils, rhizospheres, and pure cultures of rhizobacteria. Zablotowicz, R. M., Locke, M. A., and Hoagland, R. E. Phytoremediation of soil and water contaminants. Washington, DC : American Chemical Society, 1997. p. 38-53. NAL Call #: QD1.A45-no.664 Abstract: Reduction of nitroaromatic compounds to their corresponding amino derivatives is one of several pathways in the degradation of nitroxenobiotics. Our studies with the nitrodiphenyl ether herbicide acifluorfen showed rapid metabolism to am inoacifluorfen followed by incorporation into unextractable soil components in both soil and rhizosphere suspensions. Aminoacifluorfen was formed more rapidly in rhizospheres compared to soil, which can be attributed to higher microbial populations, espec ially of Gram-negative bacteria. We identified several strains of Pseudomonas fluorescens that possess nitroreductase activity capable of converting acifluorfen to aminoacifluorfen. Factors affecting acifluorfen nitroreductase activity in pure cultures an d cell-free extracts, and other catabolic transformations of acifluorfen, ether bond cleavage, are discussed. Plant rhizospheres should be conducive for aromatic nitroreduction. Nitroreduction by rhizobacteria is an important catabolic pathway for the ini tial degradation of various nitroherbicides and other nitroaromatic compounds in soils under Reduction of nitroaromatic compounds to their corresponding amino derivatives is one of several pathways in the degradation of nitroxenobiotics. Our studies with the nitrodiphenyl ether herbicide acifluorfen showed rapid metabolism to am inoacifluorfen followed by incorporation into unextractable soil components in both soil and rhizosphere suspensions. Aminoacifluorfen was formed more rapidly in rhizospheres compared to soil, which can be attributed to higher microbial populations, espec ially of Gram-negative bacteria. We identified several strains of Pseudomonas fluorescens that possess nitroreductase activity capable of converting acifluorfen to aminoacifluorfen. Factors affecting acifluorfen nitroreductase activity in pure cultures an d cell-free extracts, and other catabolic transformations of acifluorfen, ether bond cleavage, are discussed. Plant rhizospheres should be conducive for aromatic nitroreduction. Nitroreduction by rhizobacteria is an important catabolic pathway for the ini tial degradation of various nitroherbicides and other nitroaromatic compounds in soils under phytoremediation management. 8. Ascorbate: a biomarker of herbicide stress in wetland plants. Lytle, T. F. and Lytle, J. S. Phytoremediation of soil and water contaminants. Washington, DC : American Chemical Society, 1997. p. 106-113. NAL Call #: QD1.A45-no.664 Abstract: In laboratory exposures of wetland plants to low herbicide levels (<0.1 micrograms/mL), some plants showed increased total ascorbic acid suggesting a stimulatory effect on ascorbic acid synthesis occurred; at higher herbicide conce ntrations (greater than or equal to 0.1 micrograms/mL) a notable decline in total ascorbic acid and increase in the oxidized form, dehydroascorbic acid occurred. Vigna luteola and Sesbania vesicaria were exposed for 7 and 21 days respectively to atrazine (0.05 to 1 microgram/mL); Spartina alterniflora 28 days at 0.1 micrograms/mL trifluralin; Hibiscus moscheutos 14 days at 0.1 and 1 microgram/mL metolachlor in fresh and brackish water. The greatest increase following low dosage occurred with S. alterniflo ra, increasing from <600 micrograms/g wet wt. total ascorbic acid to >1000 micrograms/g. Ascorbic acid may be a promising biomarker of estuarine plants exposed to herbicide runoff; stimulation of ascorbic acid synthesis may enable some wetland plant s used in phytoremediation to cope with low levels of these compounds. In laboratory exposures of wetland plants to low herbicide levels (<0.1 micrograms/mL), some plants showed increased total ascorbic acid suggesting a stimulatory effect on ascorbic acid synthesis occurred; at higher herbicide conce ntrations (greater than or equal to 0.1 micrograms/mL) a notable decline in total ascorbic acid and increase in the oxidized form, dehydroascorbic acid occurred. Vigna luteola and Sesbania vesicaria were exposed for 7 and 21 days respectively to atrazine (0.05 to 1 microgram/mL); Spartina alterniflora 28 days at 0.1 micrograms/mL trifluralin; Hibiscus moscheutos 14 days at 0.1 and 1 microgram/mL metolachlor in fresh and brackish water. The greatest increase following low dosage occurred with S. alterniflo ra, increasing from <600 micrograms/g wet wt. total ascorbic acid to >1000 micrograms/g. Ascorbic acid may be a promising biomarker of estuarine plants exposed to herbicide runoff; stimulation of ascorbic acid synthesis may enable some wetland plant s used in phytoremediation to cope with low levels of these compounds. 9. Atmospheric nitrogenous compounds and ozone--is NO(x) fixation by plants a possible solution. Wellburn, A. R. New phytol. 139: 1 pp. 5-9. (May 1998). NAL Call #: 450-N42 Descriptors: ozoneair-pollution nitrogen-dioxide nitric-oxide air-quality tolerancebioremediationacclimatizationnutrient-sources nutrient-uptake plantscultivarsgenetic-variation literature-reviews 10. Atrazine degradation in pesticide-contaminated soils: phytoremediation potential. Kruger, E. L., Anhalt, J. C., Sorenson, D., Nelson, B., Chouhy, A. L., Anderson, T. A., and Coats, J. R. Phytoremediation of soil and water contaminants. Washington, DC : American Chemical Society, 1997. p. 54-64. NAL Call #: QD1.A45-no. 664 Abstract: Studies were conducted in the laboratory to determine the fate of atrazine in pesticide-contaminated soils from agrochemical dealer sites. No significant differences in atrazine concentrations occurred in soils treated with atrazine i ndividually or combinations with metolachlor and trifluralin. In a screening study carried out in soils from four agrochemical dealer sites, rapid mineralization of atrazine occurred in three out of eight soils tested, with the greatest amount occurring i n Bravo rhizosphere soil (35% of the applied atrazine after 9 weeks). Suppression of atrazine mineralization in the Bravo rhizosphere soil did not occur with the addition of high concentrations of herbicide mixtures, but instead was increased. Plants had a positive impact on dissipation of aged Studies were conducted in the laboratory to determine the fate of atrazine in pesticide-contaminated soils from agrochemical dealer sites. No significant differences in atrazine concentrations occurred in soils treated with atrazine i ndividually or combinations with metolachlor and trifluralin. In a screening study carried out in soils from four agrochemical dealer sites, rapid mineralization of atrazine occurred in three out of eight soils tested, with the greatest amount occurring i n Bravo rhizosphere soil (35% of the applied atrazine after 9 weeks). Suppression of atrazine mineralization in the Bravo rhizosphere soil did not occur with the addition of high concentrations of herbicide mixtures, but instead was increased. Plants had a positive impact on dissipation of aged atrazine in soil, with significantly less atrazine extractable from Kochia-vegetated soils than from nonvegetated soils. 11. Bacterial inoculants of forage grasses that enhance degradation of 2-chlorobenzoic acid in soil. Siciliano, S. D. and Germida, J. J. Environ toxicol chem. 16: 6 pp. 1098-1104. (June 1997). NAL Call #: QH545.A1E58 Descriptors: polluted-soils bioremediationAbstract: Biological remediation of contaminated soil is an effective method of reducing risk to human and ecosystem health. Bacteria and plants might be used to enhance remediation of soil pollutants in situ. This study assessed the potential of bacteria (12 isolates), plants (16 forage grasses), and plant-bacteria associations (selected pairings) to remediate 2-chlorobenzoic acid (2CBA)-contaminated soil. Initially, grass viability was assessed in 2CBA-contaminated soil. Soil was contaminated wi th 2CBA, forage grasses were grown under growth chamber conditions for 42 or 60 d, and the 2CBA concentration in soil was determined by gas chromatography. Only five of 16 forage grasses grew in 2CBA-treated (816 mg/kg) soil. Growth of Bromus inermis had no effect on 2CBA concentration, whereas Agropyron intermedium, B. biebersteinii, A. riparum, and Elymus dauricus decreased 2CBA relative to nonplanted control soil by 32 to 42%. The 12 bacteria isolates were screened for their ability to promote the germ ination of the five grasses in 2CBA-contaminated soil. Inoculation of A. riparum with Pseudomonas aeruginos

Phytoremediation : 植物による環境/土壌浄化

We performed a comprehensive approach to determine the proteome of Saccharomyces cerevisiae mitochondria. The proteins of highly pure yeast mitochondria were separated by several independent methods and analyzed by tandem MS. From >20 million MS spectra, 750 different proteins were identified, indicating an involvement of mitochondria in numerous cellular processes. All known components of the oxidative phosphorylation machinery, the tricarboxylic acid cycle, and the stable mitochondria-encoded proteins were found. Based on the mitochondrial proteins described in the literature so far, we calculate that the identified proteins represent ≈90% of all mitochondrial proteins. The function of a quarter of the identified proteins is unknown. The mitochondrial proteome will provide an important database for the analysis of new mitochondrial and mitochondria-associated functions and the characterization of mitochondrial diseases.

https://www.pnas.org/content/pnas/100/23/13207.full.pdf

The proteome of Saccharomyces cerevisiae mitochondria

Mitochondria contain approximately 1,000 different proteins, most of which are imported from the cytosol. Two import pathways that direct proteins into the mitochondrial inner membrane and matrix have been known for many years. The identification of numerous new transport components in recent proteomic studies has led to novel mechanistic insight into these pathways and the discovery of new import pathways into the outer membrane and intermembrane space. Protein translocases do not function as independent units but are integrated into dynamic networks and are connected to machineries that function in bioenergetics, mitochondrial morphology and coupling to the endoplasmic reticulum.

/pdf/mitochondrial-protein-import-from-proteomics-to-functional-9uwj6vc4l7.pdf

Mitochondrial protein import: from proteomics to functional mechanisms

An Arabidopsis mitochondrial proteome project was started for a comprehensive investigation of mitochondrial functions in plants. Mitochondria were prepared from Arabidopsis stems and leaves or from Arabidopsis suspension cell cultures, and the purity of the generated fractions was tested by the resolution of organellar protein complexes applying two-dimensional blue-native/N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine (Tricine) sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Arabidopsis mitochondrial proteome was analyzed by two-dimensional isoelectric focusing/ Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 650 different proteins in a pI range of pH 3 to 10 were separated on single gels. Solubilization conditions, pH gradients for isoelectric focusing, and gel staining procedures were varied, and the number of separable proteins increased to about 800. Fifty-two protein spots were identified by immunoblotting, direct protein sequencing, and mass spectrometry. The characterized proteins cooperate in various processes, such as respiration, citric acid cycle, amino acid and nucleotide metabolism, protection against O2, mitochondrial assembly, molecular transport, and protein biosynthesis. More than 20% of the identified proteins were not described previously for plant mitochondria, indicating novel mitochondrial functions. The map of the Arabidopsis mitochondrial proteome should be useful for the analysis of knockout mutants concerning nuclear-encoded mitochondrial genes. Considerations of the total complexity of the Arabidopsis mitochondrial proteome are discussed. The data from this investigation will be made available at http://www.gartenbau.uni-hannover.de/genetik/AMPP.

http://www.plantphysiol.org/content/plantphysiol/127/4/1694.full.pdf

Proteomic Approach to Identify Novel Mitochondrial Proteins in Arabidopsis

The translocase of the outer mitochondrial membrane (TOM) complex is a preprotein translocase that mediates transport of nuclear-encoded mitochondrial proteins across the outer mitochondrial membrane. Here we report the purification of this protein complex from Arabidopsis. On blue-native gels the Arabidopsis TOM complex runs at 230 kD and can be dissected into subunits of 34, 23, 21, 8, 7, and 6 kD. The identity of four subunits could be determined by immunoblotting and/or direct protein sequencing. The 21- and the 23-kD subunits exhibit significant sequence homology to the TOM20 preprotein receptor from other organisms. Analysis by two-dimensional isoelectric focusing/Tricine sodium dodecyl sulfide-polyacrylamide gel electrophoresis revealed the presence of further forms for Arabidopsis TOM20. All TOM20 proteins comprise a large cytoplasmically exposed hydrophilic domain, which is degraded upon trypsination of intact mitochondria. Clones encoding four different forms of Arabidopsis TOM20 were identified and sequenced. The deduced amino acid sequences are rather conserved in the N-terminal half and in the very C-terminal part, but include a highly variable glycine-rich region close to the C terminus. Implications on the function of plant TOM complexes are discussed. Based on peptide and nucleic acid sequence data, the primary structure for Arabidopsis TOM40 is presented.

Purification and Characterization of the Preprotein Translocase of the Outer Mitochondrial Membrane from Arabidopsis. Identification of Multiple Forms of TOM20

We report a subdivision of the mitochondrial proteome into defined sets of proteins, which is based on the combination of three different gel electrophoresis procedures. First, Blue-native polyacrylamide gel electrophoresis is employed to separate mitochondrial protein complexes. The protein complexes are electroeluted and completely detached from Coomasssie blue. Subsequently the subunits of the protein complexes are separated by isoelectric focusing and finally by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The resolution capacity of the procedure is demonstrated for the ATP synthase complex, the cytochrome c reductase complex and the preprotein translocase of the outer mitochondrial membrane (the TOM complex). The method allows the separation of isoforms of subunits forming part of protein complexes, whose occurrence seems to be rather a rule than an exception in higher eukaryotes. Furthermore, extremely hydrophobic proteins are detectable on the gels.

Biochemical dissection of the mitochondrial proteome from Arabidopsis thaliana by three-dimensional gel electrophoresis.

Identification of novel subunits of the TOM complex from Arabidopsis thaliana

It is a difficult task to isolate mitochondria from green tissue, especially from the widely used model plant Arabidopsis thaliana. However, for several physiological queries very pure and physiologically intact mitochondria are needed. From three plant species, Pisum sativum L., Nicotiana tabacum L. and Arabidopsis thaliana HEYNH., mitochondria were isolated according to different purification protocols described before or by an own optimised protocol. The methods involved differential centrifugation and density gradient centrifugation on self-generating gradients or step-gradients of Percoll. The quality of the purified mitochondria were tested by different methods to prove intactness of the membrane system, the physiological activity and the contamination by other organelles. In summary, it was possible to isolate mitochondria from the three different plant species according to all criteria investigated. However, the reliability of the organelle preparations and the purity and intactness varied between the methods. To demonstrate the quality of the purified mitochondria with respect to different traits a combination of several tests is recommendable. Determination of chlorophyll contents and enzyme activities, functional analysis of the respiratory chain, and the use of the powerful technique of Blue-native/Tricine SDS polyacrylamide gel electrophoresis are necessary to fully characterize the mitochondria. Especially combining a visual result by comparing the separation of protein complexes in gels with physiological measurements seems to be very useful for a comprehensive characterization of the organelle preparation.

Wolf Werhahn

Papers

Proteomic Approach to Identify Novel Mitochondrial Proteins in Arabidopsis

Purification and Characterization of the Preprotein Translocase of the Outer Mitochondrial Membrane from Arabidopsis. Identification of Multiple Forms of TOM20

Biochemical dissection of the mitochondrial proteome from Arabidopsis thaliana by three-dimensional gel electrophoresis.

Identification of novel subunits of the TOM complex from Arabidopsis thaliana

How to Document the Purity of Mitochondria Prepared from Green Tissue of Pea, Tobacco and Arabidopsis thaliana