David M. Rhoads

Mitochondrial Reactive Oxygen Species. Contribution to Oxidative Stress and Interorganellar Signaling

The inner membrane of a plant mitochondrion contains the mitochondrial electron transport chain (mtETC), consisting of protein complexes that use an energy source-derived reductant to form a proton gradient across the membrane. This proton gradient drives ATP synthesis, a primary mitochondrial

Identification of a region of the Arabidopsis AtAOX1a promoter necessary for mitochondrial retrograde regulation of expression.

Chemical inhibition of the mitochondrial electron transport chain (mtETC) by antimycin A (AA) or the TCA cycle by monofluoroacetate (MFA) causes increased expression of nucleus-encoded alternative oxidase (AOX) genes in plants. In order to better understand the mechanisms of this mitochondrial retrograde regulation (MRR) of gene expression, constructs containing deleted and mutated versions of a promoter region of the Arabidopsis thaliana AOX1a gene (AtAOX1a) controlling expression of the coding region of the enhanced firefly luciferase gene were employed to identify regions of the AtAOX1a promoter important for induction in response to mtETC or TCA cycle inhibition. Transient transformation coupled with in vitro and in vivo assays as well as plants containing transgenes with truncated promoter regions were used to identify a 93 base pair portion of the promoter, termed the MRR region, that was necessary for induction. Further mutational analyses showed that most of the 93 bp MRR region is important for both AA and MFA induction. Sub-regions within the MRR region that are especially important for strong induction by both AA or MFA were identified. Specific mutations in a W-box and Dof motifs in the MRR region indicate that these specific motifs are not important for induction. Recent evidence suggests that MRR of AOX genes following inhibition of the mtETC is via a separate signaling pathway from MRR resulting from metabolic shifts, such as those that result from MFA treatment. Our data suggest that these signaling pathways share regulatory regions in the AtAOX1a promoter. Arabidopsis proteins interacted specifically with a probe containing the MRR region, as shown by electrophoretic mobility shift assays and Southwestern blotting. These interactions were eliminated under reducing conditions.

A reporter gene system used to study developmental expressionof alternative oxidase and isolate mitochondrial retrograde regulationmutants in Arabidopsis

Perturbation of mitochondrial function causes altered nuclear gene expression in plants. To study this response, called mitochondrial retrograde regulation, and developmental gene expression, a transgenic Arabidopsis thaliana (Col-0) line containing a firefly luciferase gene controlled by a promoter region of the Arabidopsis alternative oxidase 1a gene (AtAOX1a) was created. The transgene and the endogenous gene were developmentally induced in young cotyledons to a level higher than in older cotyledons and leaves. Analysis of transgene expression suggests that this is true for emerging leaves as well. Antimycin A (AA), a mitochondrial electron transport chain inhibitor, and monofluroacetate (MFA), a TCA cycle inhibitor, induced expression of the transgene and the endogenous gene in parallel. The following comparative responses of the transgene to inhibitors were observed: (a) the response in cotyledons to AA treatment differed greatly in magnitude from the response in leaves; (b) the induction kinetics in cotyledons following MFA treatment differed greatly from the kinetics in leaves; and (c) the induction kinetics following MFA treatment differed from the kinetics of AA in both leaves and cotyledons. The transgenic line was used in a genetic screen to isolate mutants with greatly decreased transgene and AtAOX1a induction in response to AA. Some of these mutant lines showed greatly decreased induction by MFA, but one did not. Taken altogether, the data provide genetic evidence that suggests that induction of the AtAOX1a gene by distinct mitochondrial perturbations are via distinct, but overlapping signaling pathways that are tissue specific.

Mitochondrial localization and putative signaling function of sucrose synthase in maize

In many organisms, an increasing number of proteins seem to play two or more unrelated roles. Here we report that maize sucrose synthase (SUS) is distributed in organelles not involved in sucrose metabolism and may have novel roles beyond sucrose degradation. Bioinformatics analysis predicts that among the three maize SUS isoforms, SH1 protein has a putative mitochondrial targeting peptide (mTP). We validated this prediction by the immunodetection of SUS in mitochondria. Analysis with isoform-specific antisera revealed that both SH1 and SUS1 are represented in mitochondria, although the latter lacks a canonical mTP. The SUS2 isoform is not detectable in mitochondria, despite its presence in the cytosol. In maize primary roots, the mitochondrion-associated SUS (mtSUS; which includes SH1 and SUS1) is present mostly in the root tip, indicating tissue-specific regulation of SUS compartmentation. Unlike the glycolytic enzymes that occur attached to the outside of mitochondria, SH1 and SUS1 are intramitochondrial. The low abundance of SUS in mitochondria, its high Km value for sucrose, and the lack of sucrose in mitochondria suggest that mtSUS plays a non-sucrolytic role. Co-immunoprecipitation studies indicate that SUS interacts with the voltage-dependent anion channel in an isoform-specific and anoxia-enhanced manner and may be involved in the regulation of solute fluxes into and out of mitochondria. In several plant species, at least one of the SUS proteins possesses a putative mTP, indicating the conservation of the noncatalytic function across plant species. Taken together, these observations suggest that SUS has a novel noncatalytic function in plant cells.

Comparison of Intact Arabidopsis thaliana Leaf Transcript Profiles during Treatment with Inhibitors of Mitochondrial Electron Transport and TCA Cycle

Plant mitochondria signal to the nucleus leading to altered transcription of nuclear genes by a process called mitochondrial retrograde regulation (MRR). MRR is implicated in metabolic homeostasis and responses to stress conditions. Mitochondrial reactive oxygen species (mtROS) are a MRR signaling component, but whether all MRR requires ROS is not established. Inhibition of the cytochrome respiratory pathway by antimycin A (AA) or the TCA cycle by monofluoroacetate (MFA), each of which initiates MRR, can increase ROS production in some plant cells. We found that for AA and MFA applied to leaves of soil-grown Arabidopsis thaliana plants, ROS production increased with AA, but not with MFA, allowing comparison of transcript profiles under different ROS conditions during MRR. Variation in transcript accumulation over time for eight nuclear encoded mitochondrial protein genes suggested operation of both common and distinct signaling pathways between the two treatments. Consequences of mitochondrial perturbations for the whole transcriptome were examined by microarray analyses. Expression of 1316 and 606 genes was altered by AA and MFA, respectively. A subset of genes was similarly affected by both treatments, including genes encoding photosynthesis-related proteins. MFA treatment resulted in more down-regulation. Functional gene category (MapMan) and cluster analyses showed that genes with expression levels affected by perturbation from AA or MFA inhibition were most similarly affected by biotic stresses such as pathogens. Overall, the data provide further evidence for the presence of mtROS-independent MRR signaling, and support the proposed involvement of MRR and mitochondrial function in plant responses to biotic stress.

Plant Mitochondrial Retrograde Regulation

Changes in plant cellular metabolism require altered gene expression. Cellular adjustments and altered gene expression also occur in response to environmental stresses. Both of these situations involve changes in mitochondrial activities. Changes in the status of the mitochondria and resulting inputs for altered nuclear gene expression are communicated to the nucleus by interorganellar signaling called mitochondrial retrograde regulation (MRR). The study of plant MRR is a young field and the mechanisms and components are just beginning to be discovered. Evidence suggests that reactive oxygen species (ROS) can be involved in plant MRR, but this does not mean that they are involved in all cases. Calcium level changes, redox changes, and changes in metabolite levels are leading candidates for nonprotein signaling components. Protein signaling components like kinases, phosphatases, and transcription factors are likely to be involved, but only the recent discovery of the involvement of transcription factor Abscisic Acid Insensitive 4 in Arabidopsis provides a specific protein example. Growing evidence indicates overlaps of MRR with other signaling pathways, including those from chloroplasts. MRR could be linked to metabolic signaling and/or ROS production, but these would still originate in mitochondria and be components of MRR. The emerging view is that plant mitochondria are stress sensors that contribute to decisions regarding cell fate during stresses and that this is conveyed to the nucleus by MRR.

Reconciliation of Sequence Data and Updated Annotation of the Genome of Agrobacterium tumefaciens C58, and Distribution of a Linear Chromosome in the Genus Agrobacterium

ABSTRACT Two groups independently sequenced the Agrobacterium tumefaciens C58 genome in 2001. We report here consolidation of these sequences, updated annotation, and additional analysis of the evolutionary history of the linear chromosome, which is apparently limited to the biovar I group of Agrobacterium.

Sucrose synthase: expanding protein function.

Sucrose synthase (SUS: EC 2.4.1.13), a key enzyme in plant sucrose catabolism, is uniquely able to mobilize sucrose into multiple pathways involved in metabolic, structural, and storage functions. Our research indicates that the biological function of SUS may extend beyond its catalytic activity. This inference is based on the following observations: a) tissue-specific, isoform-dependent and metabolically-regulated association of SUS with mitochondria and b) isoform-specific and anoxia-responsive interaction of SUS with the voltage-dependent anion channel (VDAC), the major outer mitochondrial membrane protein. More recent work shows that both VDAC and SUS are also localized to the nucleus in maize seedling tissues. Their intricate regulation under anoxia indicates that these two proteins may have a role in inter-compartmental signaling.

Mitochondria-Nucleus Interactions: Evidence for Mitochondrial Retrograde Communication in Plant Cells

There is increasing awareness of the intricate communication networks and signal transduction pathways that link plant organelles. It appears that in some situations altered expression of specific nuclear genes is directed by other organelles. This is referred to as retrograde regulation of nuclear gene expression and is presumably dependent upon some form of retrograde communication (signaling) from the other organelles to the nucleus. Relatively little is known about the mechanisms involved in mitochondrial retrograde regulation of nuclear gene expression in plants. Nonetheless, experimental results from yeast, mammalian and plant cells indicate that mitochondria participate in the sensing of developmental cues, as well as biotic and abiotic stresses, and that this sensing then initiates retrograde regulation of nuclear gene expression. Here we present the current state of research regarding the potential roles and mechanisms of mitochondrial retrograde regulation in higher plants.

[47]Assays for characterizing URF 13, the pathotoxin and methomyl receptor of cms-T maize

Publisher Summary This chapter explains assays for characterizing URF 13, the Pathotoxin and Methomyl receptor of cms- T maize. Maize carrying the Texas cytoplasm is male sterile. Cytoplasmic male sterility (CMS) in cms-T maize is characterized by pollen abortion and failure to exert anthers, but female fertility is unaffected. Maize containing the cms-T cytoplasm is used extensively in hybrid seed production as it eliminates the need for manual emasculation. A 13-kDa protein—URF13—is the product of a mitochondrial gene called T-urf13, which is specific to cms-T maize and arises from a novel series of rearrangements within the mitochondrial genome. URF13 is an integral membrane protein that is located in the inner mitochondrial membrane. Escherichia coli cells expressing URF13 are also sensitive to T-toxin. T-toxin interacts with isolated cms-T mitochondria: cell respiration is inhibited, spheroplasts swell, and ion leakage is induced. Methomyl is often used in assays and in selective screens for the presence of functional URF13 as it is readily available (as Lannate), whereas T-toxin is more difficult to obtain. The effects of T-toxins or methomyl on cms-T mitochondria have led to the hypothesis that the interaction between these compounds and URF13 results in the formation of hydrophilic pores that permeabilize the inner mitochondrial membrane.