The Transcription Factor MYB29 Is a Regulator of ALTERNATIVE OXIDASE1a.

Plants sense and integrate a variety of signals from the environment through different interacting signal transduction pathways that involve hormones and signaling molecules. Using ALTERNATIVE OXIDASE1a (AOX1a) gene expression as a model system of retrograde or stress signaling between mitochondria and the nucleus, MYB DOMAIN PROTEIN29 (MYB29) was identified as a negative regulator (regulator of alternative oxidase1a 7 [rao7] mutant) in a genetic screen of Arabidopsis (Arabidopsis thaliana). rao7/myb29 mutants have increased levels of AOX1a transcript and protein compared to wild type after induction with antimycin A. A variety of genes previously associated with the mitochondrial stress response also display enhanced transcript abundance, indicating that RAO7/MYB29 negatively regulates mitochondrial stress responses in general. Meta-analysis of hormone-responsive marker genes and identification of downstream transcription factor networks revealed that MYB29 functions in the complex interplay of ethylene, jasmonic acid, salicylic acid, and reactive oxygen species signaling by regulating the expression of various ETHYLENE RESPONSE FACTOR and WRKY transcription factors. Despite an enhanced induction of mitochondrial stress response genes, rao7/myb29 mutants displayed an increased sensitivity to combined moderate light and drought stress. These results uncover interactions between mitochondrial retrograde signaling and the regulation of glucosinolate biosynthesis, both regulated by RAO7/MYB29. This common regulator can explain why perturbation of the mitochondrial function leads to transcriptomic responses overlapping with responses to biotic stress.

Characterization of a novel ß-barrel protein (AtOM47) from the mitochondrial outer membrane of Arabidopsis thaliana.

In plant cells, mitochondria are major providers of energy and building blocks for growth and development as well as abiotic and biotic stress responses. They are encircled by two lipid membranes containing proteins that control mitochondrial function through the import of macromolecules and metabolites. Characterization of a novel ß-barrel protein, OUTER MEMBRANE PROTEIN 47 (OM47), unique to the green lineage and related to the voltage-dependent anion channel (VDAC) protein family, showed that OM47 can complement a VDAC mutant in yeast. Mutation of OM47 in Arabidopsis thaliana by T-DNA insertion had no effect on the import of proteins, such as the ß-barrel proteins translocase of the outer membrane 40 (TOM40) or sorting and assembly machinery 50 (SAM50), into mitochondria. Molecular and physiological analyses revealed a delay in chlorophyll breakdown, higher levels of starch, and a delay in the induction of senescence marker genes in the mutant lines. While there was a reduction of >90% in OM47 protein in mitochondria isolated from 3-week-old om47 mutants, in mitochondria isolated from 8-week-old plants OM47 levels were similar to that of the wild type. This recovery was achieved by an up-regulation of OM47 transcript abundance in the mutants. Combined, these results highlight a role in leaf senescence for this plant-specific ß-barrel protein, probably mediating the recovery and recycling of chloroplast breakdown products by transporting metabolic intermediates into and out of mitochondria.

Inactivation of Mitochondrial Complex I Induces the Expression of a Twin Cysteine Protein that Targets and Affects Cytosolic, Chloroplastidic and Mitochondrial Function.

At12Cys-1 (At5g64400) and At12Cys-2 (At5g09570) are two closely related isogenes that encode small, twin cysteine proteins, typically located in mitochondria. At12Cys-2 transcript is induced in a variety of mutants with disrupted mitochondrial proteins, but an increase in At12Cys protein is only detected in mutants with reduced mitochondrial complex I abundance. Induction of At12Cys protein in mutants that lack mitochondrial complex I is accompanied by At12Cys protein located in mitochondria, chloroplasts, and the cytosol. Biochemical analyses revealed that even single gene deletions, i.e., At12cys-1 or At12cys-2, have an effect on mitochondrial and chloroplast functions. However, only double mutants, i.e., At12cys-1:At12cys-2, affect the abundance of protein and mRNA transcripts encoding translation elongation factors as well as rRNA abundance. Blue native PAGE showed that At12Cys co-migrated with mitochondrial supercomplex I + III. Likewise, deletion of both At12cys-1 and At12cys-2 genes, but not single gene deletions, results in enhanced tolerance to drought and light stress and increased anti-oxidant capacity. The induction and multiple localization of At12Cys upon a reduction in complex I abundance provides a mechanism to specifically signal mitochondrial dysfunction to the cytosol and then beyond to other organelles in the cell.

The mitochondrial outer membrane AAA ATPase AtOM66 affects cell death and pathogen resistance in Arabidopsis thaliana.

One of the most stress-responsive genes encoding a mitochondrial protein in Arabidopsis (At3g50930) has been annotated as AtBCS1 (cytochrome bc1 synthase 1), but was previously functionally uncharacterised. Here, we show that the protein encoded by At3g50930 is present as a homo-multimeric protein complex on the outer mitochondrial membrane and lacks the BCS1 domain present in yeast and mammalian BCS1 proteins, with the sequence similarity restricted to the AAA ATPase domain. Thus we propose to re-annotate this protein as AtOM66 (Outer Mitochondrial membrane protein of 66 kDa). While transgenic plants with reduced AtOM66 expression appear to be phenotypically normal, AtOM66 over-expression lines have a distinct phenotype, showing strong leaf curling and reduced starch content. Analysis of mitochondrial protein content demonstrated no detectable changes in mitochondrial respiratory complex protein abundance. Consistent with the stress inducible expression pattern, over-expression lines of AtOM66 are more tolerant to drought stress but undergo stress-induced senescence earlier than wild type. Genome-wide expression analysis revealed a constitutive induction of salicylic acid-related (SA) pathogen defence and cell death genes in over-expression lines. Conversely, expression of SA marker gene PR-1 was reduced in atom66 plants, while jasmonic acid response genes PDF1.2 and VSP2 have increased transcript abundance. In agreement with the expression profile, AtOM66 over-expression plants show increased SA content, accelerated cell death rates and are more tolerant to the biotrophic pathogen Pseudomonas syringae, but more susceptible to the necrotrophic fungus Botrytis cinerea. In conclusion, our results demonstrate a role for AtOM66 in cell death and amplifying SA signalling.

A Functional Antagonistic Relationship between Auxin and Mitochondrial Retrograde Signaling Regulates Alternative Oxidase1a Expression in Arabidopsis.

The perception and integration of stress stimuli with that of mitochondrion function are important during periods of perturbed cellular homeostasis. In a continuous effort to delineate these mitochondrial/stress-interacting networks, forward genetic screens using the mitochondrial stress response marker alternative oxidase 1a (AOX1a) provide a useful molecular tool to identify and characterize regulators of mitochondrial stress signaling (referred to as regulators of alternative oxidase 1a [RAOs] components). In this study, we reveal that mutations in genes coding for proteins associated with auxin transport and distribution resulted in a greater induction of AOX1a in terms of magnitude and longevity. Three independent mutants for polarized auxin transport, rao3/big, rao4/pin-formed1, and rao5/multidrug-resistance1/abcb19, as well as the Myb transcription factor rao6/asymmetric leaves1 (that displays altered auxin patterns) were identified and resulted in an acute sensitivity toward mitochondrial dysfunction. Induction of the AOX1a reporter system could be inhibited by the application of auxin analogs or reciprocally potentiated by blocking auxin transport. Promoter activation studies with AOX1a::GUS and DR5::GUS lines further confirmed a clear antagonistic relationship between the spatial distribution of mitochondrial stress and auxin response kinetics, respectively. Genome-wide transcriptome analyses revealed that mitochondrial stress stimuli, such as antimycin A, caused a transient suppression of auxin signaling and conversely, that auxin treatment repressed a part of the response to antimycin A treatment, including AOX1a induction. We conclude that mitochondrial stress signaling and auxin signaling are reciprocally regulated, balancing growth and stress response(s).

Organellar oligopeptidase (OOP) provides a complementary pathway for targeting peptide degradation in mitochondria and chloroplasts.

Both mitochondria and chloroplasts contain distinct proteolytic systems for precursor protein processing catalyzed by the mitochondrial and stromal processing peptidases and for the degradation of targeting peptides catalyzed by presequence protease. Here, we have identified and characterized a component of the organellar proteolytic systems in Arabidopsis thaliana, the organellar oligopeptidase, OOP (At5g65620). OOP belongs to the M3A family of peptide-degrading metalloproteases. Using two independent in vivo methods, we show that the protease is dually localized to mitochondria and chloroplasts. Furthermore, we localized the OPP homolog At5g10540 to the cytosol. Analysis of peptide degradation by OOP revealed substrate size restriction from 8 to 23 aa residues. Short mitochondrial targeting peptides (presequence of the ribosomal protein L29 and presequence of 1-aminocyclopropane-1-carboxylic acid deaminase 1) and N- and C-terminal fragments derived from the presequence of the ATPase beta subunit ranging in size from 11 to 20 aa could be degraded. MS analysis showed that OOP does not exhibit a strict cleavage pattern but shows a weak preference for hydrophobic residues (F/L) at the P1 position. The crystal structures of OOP, at 1.8-1.9 Å, exhibit an ellipsoidal shape consisting of two major domains enclosing the catalytic cavity of 3,000 Å(3). The structural and biochemical data suggest that the protein undergoes conformational changes to allow peptide binding and proteolysis. Our results demonstrate the complementary role of OOP in targeting-peptide degradation in mitochondria and chloroplasts.

Coordinating metabolite changes with our perception of plant abiotic stress responses: emerging views revealed by integrative-omic analyses.

Metabolic configuration and adaptation under a range of abiotic stresses, including drought, heat, salinity, cold, and nutrient deprivation, are subjected to an intricate span of molecular pathways that work in parallel in order to enhance plant fitness and increase stress tolerance. In recent years, unprecedented advances have been made in identifying and linking different abiotic stresses, and the current challenge in plant molecular biology is deciphering how the signaling responses are integrated and transduced throughout metabolism. Metabolomics have often played a fundamental role in elucidating the distinct and overlapping biochemical changes that occur in plants. However, a far greater understanding and appreciation of the complexity in plant metabolism under specific stress conditions have become apparent when combining metabolomics with other-omic platforms. This review focuses on recent advances made in understanding the global changes occurring in plant metabolism under abiotic stress conditions using metabolite profiling as an integrated discovery platform.