RAC/ROP proteins (r-related GTPases of plants) are plant-specific small G proteins that function as molecular switches within elementary signal transduction pathways, including the regulation of reactive oxygen species (ROS) generation during early microbial infection via the activation of NADPH oxidase homologs of plants termed RBOH (for respiratory burst oxidase homolog). We investigated the role of Medicago truncatula Jemalong A17 small GTPase MtROP9, orthologous to Medicago sativa Rac1, via an RNA interference silencing approach. Composite M. truncatula plants (MtROP9i) whose roots have been transformed by Agrobacterium rhizogenes carrying the RNA interference vector were generated and infected with the symbiotic arbuscular mycorrhiza fungus Glomus intraradices and the rhizobial bacterium Sinorhizobium meliloti as well as with the pathogenic oomycete Aphanomyces euteiches. MtROP9i transgenic lines showed a clear growth-reduced phenotype and revealed neither ROS generation nor MtROP9 and MtRBOH gene expression after microbial infection. Coincidently, antioxidative compounds were not induced in infected MtROP9i roots, as documented by differential proteomics (two-dimensional differential gel electrophoresis). Furthermore, MtROP9 knockdown clearly promoted mycorrhizal and A. euteiches early hyphal root colonization, while rhizobial infection was clearly impaired. Infected MtROP9i roots showed, in part, extremely swollen noninfected root hairs and reduced numbers of deformed nodules. S. meliloti nodulation factor treatments of MtROP9i led to deformed root hairs showing progressed swelling of its upper regions or even of the entire root hair and spontaneous constrictions but reduced branching effects occurring only at swollen root hairs. These results suggest a key role of Rac1 GTPase MtROP9 in ROS-mediated early infection signaling.

The succinate dehydrogenase complex (complex II) is a highly conserved protein complex composed of the SDH1 to SDH4 subunits in bacteria and in the mitochondria of animals and fungi. The reason for the occurrence of up to four additional subunits in complex II of plants, termed SDH5 to SDH8, so far is a mystery. Here, we present a biochemical approach to investigate the internal subunit arrangement of Arabidopsis (Arabidopsis thaliana) complex II. Using low-concentration detergent treatments, the holo complex is dissected into subcomplexes that are analyzed by a three-dimensional gel electrophoresis system. Protein identifications by mass spectrometry revealed that the largest subcomplex (IIa) represents the succinate dehydrogenase domain composed of SDH1 and SDH2. Another subcomplex (IIb) is composed of the SDH3, SDH4, SDH6, and SDH7 subunits. All four proteins include transmembrane helices and together form the membrane anchor of complex II. Sequence analysis revealed that SDH3 and SDH4 lack helices conserved in other organisms. Using homology modeling and phylogenetic analyses, we present evidence that SDH6 and SDH7 substitute missing sequence stretches of SDH3 and SDH4 in plants. Together with SDH5, which is liberated upon dissection of complex II into subcomplexes, SDH6 and SDH7 also add some hydrophilic mass to plant complex II, which possibly inserts further functions into this smallest protein complex of the oxidative phosphorylation system (which is not so small in plants).Succinate dehydrogenase (EC 1.3.5.1) is of central importance for energy metabolism in bacteria and mitochondria of eukaryotic cells. In mitochondria, it represents the complex II of the oxidative phosphorylation (OXPHOS) system (Hatefi, 1985) and is located in the inner mitochondrial membrane. Complex II participates in two major mitochondrial processes: the tricarboxylic acid cycle as well as the mitochondrial electron transfer chain (mETC). In the tricarboxylic acid cycle, it catalyzes the conversion of succinate into fumarate. Electrons originating from this reaction are inserted into the mETC and used for the reduction of ubiquinone to ubiquinol. In contrast to other protein complexes of the OXPHOS system, complex II does not contribute to the proton gradient across the inner mitochondrial membrane.The overall structure of the succinate dehydrogenase complex is remarkably conserved in bacteria, animals, and fungi, as revealed by biochemical investigations and x-ray crystallography (Yankovskaya et al., 2003;Oyedotun and Lemire, 2004;Sun et al., 2005;Huang et al., 2006;Iverson, 2013). It is about 120 kD in size and composed of four subunits designated SDH1, SDH2, SDH3, and SDH4 (also named SDHA to SDHD in some bacteria). SDH1 is the largest subunit and includes the succinate-binding site. Electrons from succinate are accepted by a covalently bound FAD group. SDH2 carries three iron-sulfur clusters that mediate electron transfer from SDH1 to the membrane domain of complex II. Together with SDH1, it constitutes the succinate dehydrog...

Containing plastids and vacuoles in addition to those organelles also found in other (heterotrophic) cells, the plant cell displays an extraordinary level of compartmentalization, largely obtained by the utilization of membranes. These membranes not only confine reaction spaces but must also facilitate cross-talk between organelles and other cell compartments. They also host important components of the plant energy metabolism, i.e., the electron transport chains of mitochondria and chloroplasts. Characterization of the proteomes of these membranes requires isolation of pure and intact organelles from plant tissues followed by subsequent purification of their respective membranes. Membrane fractions are then amenable for further analyses using gel electrophoresis procedures or gel-free proteomic approaches. Here, we describe the preparation of intact mitochondria from Arabidopsis thaliana cell-culture, the isolation of outer and inner mitochondrial membranes and downstream proteomic applications for analyzing their membrane protein content.