Upon disturbance of their function by stress, mitochondria can signal to the nucleus to steer the expression of responsive genes. This mitochondria-to-nucleus communication is often referred to as mitochondrial retrograde regulation (MRR). Although reactive oxygen species and calcium are likely candidate signaling molecules for MRR, the protein signaling components in plants remain largely unknown. Through meta-analysis of transcriptome data, we detected a set of genes that are common and robust targets of MRR and used them as a bait to identify its transcriptional regulators. In the upstream regions of these mitochondrial dysfunction stimulon (MDS) genes, we found a cis-regulatory element, the mitochondrial dysfunction motif (MDM), which is necessary and sufficient for gene expression under various mitochondrial perturbation conditions. Yeast one-hybrid analysis and electrophoretic mobility shift assays revealed that the transmembrane domain–containing NO APICAL MERISTEM/ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR/CUP-SHAPED COTYLEDON transcription factors (ANAC013, ANAC016, ANAC017, ANAC053, and ANAC078) bound to the MDM  cis-regulatory element. We demonstrate that ANAC013 mediates MRR-induced expression of the MDS genes by direct interaction with the MDM  cis-regulatory element and triggers increased oxidative stress tolerance. In conclusion, we characterized ANAC013 as a regulator of MRR upon stress in Arabidopsis thaliana.

The role of plant mitochondrial outer membrane proteins in the process of preprotein import was investigated, as some of the principal components characterized in yeast have been shown to be absent or evolutionarily distinct in plants. Three outer membrane proteins of Arabidopsis thaliana mitochondria were studied: TOM20 (translocase of the outer mitochondrial membrane), METAXIN, and mtOM64 (outer mitochondrial membrane protein of 64 kD). A single functional Arabidopsis TOM20 gene is sufficient to produce a normal multisubunit translocase of the outer membrane complex. Simultaneous inactivation of two of the three TOM20 genes changed the rate of import for some precursor proteins, revealing limited isoform subfunctionalization. Inactivation of all three TOM20 genes resulted in severely reduced rates of import for some but not all precursor proteins. The outer membrane protein METAXIN was characterized to play a role in the import of mitochondrial precursor proteins and likely plays a role in the assembly of β-barrel proteins into the outer membrane. An outer mitochondrial membrane protein of 64 kD (mtOM64) with high sequence similarity to a chloroplast import receptor was shown to interact with a variety of precursor proteins. All three proteins have domains exposed to the cytosol and interacted with a variety of precursor proteins, as determined by pull-down and yeast two-hybrid interaction assays. Furthermore, inactivation of one resulted in protein abundance changes in the others, suggesting functional redundancy. Thus, it is proposed that all three components directly interact with precursor proteins to participate in early stages of mitochondrial protein import.

Seventeen loci encode proteins of the preprotein and amino acid transporter family in Arabidopsis (Arabidopsis thaliana). Some of these genes have arisen from recent duplications and are not in annotated duplicated regions of the Arabidopsis genome. In comparison to a number of other eukaryotic organisms, this family of proteins has greatly expanded in plants, with 24 loci in rice (Oryza sativa). Most of the Arabidopsis and rice genes are orthologous, indicating expansion of this family before monocot and dicot divergence. In vitro protein uptake assays, in vivo green fluorescent protein tagging, and immunological analyses of selected proteins determined either mitochondrial or plastidic localization for 10 and six proteins, respectively. The protein encoded by At5g24650 is targeted to both mitochondria and chloroplasts and, to our knowledge, is the first membrane protein reported to be targeted to mitochondria and chloroplasts. Three genes encoded translocase of the inner mitochondrial membrane (TIM)17-like proteins, three TIM23-like proteins, and three outer envelope protein16-like proteins in Arabidopsis. The identity of Arabidopsis TIM22-like proteins is most likely a protein encoded by At3g10110/At1g18320, based on phylogenetic analysis, subcellular localization, and complementation of a yeast (Saccharomyces cerevisiae) mutant and coexpression analysis. The lack of a preprotein and amino acid transporter domain in some proteins, localization in mitochondria, plastids, or both, variation in gene structure, and the differences in expression profiles indicate that the function of this family has diverged in plants beyond roles in protein translocation.

Diurnal regulation of transcripts encoding proteins located in mitochondria, plastids, and peroxisomes is important for adaptation of organelle biogenesis and metabolism to meet cellular requirements. We show this regulation is related to diurnal changes in promoter activities and the presence of specific cis-acting regulatory elements in the proximal promoter region [TGGGC(C/T)], previously defined as site II elements, and leads to diurnal changes in organelle protein abundances. These site II elements can act both as activators or repressors of transcription, depending on the night/day period and on the number and arrangement of site II elements in the promoter tested. These elements bind to the TCP family of transcriptions factors in Arabidopsis thaliana, which nearly all display distinct diurnal patterns of cycling transcript abundance. TCP2, TCP3, TCP11, and TCP15 were found to interact with different components of the core circadian clock in both yeast two-hybrid and direct protein-protein interaction assays, and tcp11 and tcp15 mutant plants showed altered transcript profiles for a number of core clock components, including LATE ELONGATED HYPOCOTYL1 and PSEUDO RESPONSE REGULATOR5. Thus, site II elements in the promoter regions of genes encoding mitochondrial, plastid, and peroxisomal proteins provide a direct mechanism for the coordination of expression for genes involved in a variety of organellar functions, including energy metabolism, with the time-of-day specific needs of the organism.

SummaryA variety of approaches were used to predict dual-targeted proteins in Arabidopsis thaliana. These predictions were experimentally tested using GFP fusions. Twelve new dual-targeted proteins were identified: five that were dual-targeted to mitochondria and plastids, six that were dual-targeted to mitochondria and peroxisomes, and one that was dual-targeted to mitochondria and the nucleus. Two methods to predict dual-targeted proteins had a high success rate: (1) combining the AraPerox database with a variety of subcellular prediction programs to identify mitochondrial-and peroxisomal-targeted proteins, and (2) using a variety of prediction programs on a biochemical pathway or process known to contain at least one dualtargeted protein. Several technical parameters need to be taken into account before assigning subcellular localization using GFP fusion proteins. The position of GFP with respect to the tagged polypeptide, the tissue or cells used to detect subcellular localization, and the portion of a candidate protein fused to GFP are all relevant to the expression and targeting of a fusion protein. Testing all gene models for a chromosomal locus is required if more than one model exists.

SUMMARYWe have identified a mitochondrial protein (RUG3) that is required for accumulation of mitochondrial respiratory chain complex I. RUG3 is related to human REGULATOR OF CHROMOSOME CONDENSATION 1 (RCC1) and Arabidopsis UV-B RESISTANCE 8 (UVR8). Although the family of RCC1-like proteins in Arabidopsis has over 20 members, UVR8 is the sole plant representative of this family to have been functionally characterized. Mitochondria from Arabidopsis plants lacking a functional RUG3 gene showed greatly reduced complex I abundance and activity. In contrast, accumulation of complexes III, IV and V of the oxidative phosphorylation system and the capacity for succinate-dependent respiration were unaffected. A comprehensive study of processes contributing to complex I biogenesis in rug3 mutants revealed that RUG3 is required for efficient splicing of the nad2 mRNA, which encodes a complex I subunit. A comparison of the formation of complex I assembly intermediates between rug3 and wild type mitochondria indicated that NAD2 enters the assembly pathway at an early stage. Remarkably, rug3 mutants displayed increased capacities for import of nucleus-encoded mitochondrial proteins into the organelle and showed moderately increased mitochondrial transcript levels. This observation is consistent with global transcript changes indicating enhanced mitochondrial biogenesis in the rug3 mutant in response to the complex I defect.

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The intramitochondrial location of putative type II NAD(P)H dehydrogenases (NDs) in Arabidopsis was investigated by measuring the ability of isolated mitochondria to take up precursor proteins generated from cDNAs using an in vitro translation system. The mature proteins of NDA1, NDA2 and NDC1 were judged to be located on the inside of the inner membrane because they were protected from protease added after the mitochondrial outer membrane had been ruptured. In contrast, NDB1, NDB2 and NDB4 were not protected from protease digestion in mitochondria with ruptured outer membranes and were deemed to be located on the outside of the inner membrane. Expression of all ND genes was measured using quantitative reverse transcription-PCR (RT-PCR) to determine transcript abundance, and compared with expression of alternative oxidase, uncoupler proteins and selected components of the oxidative phosphorylation complexes. NDA1 and NDB2 were the most prominently expressed members in a variety of tissues, and were up-regulated in the early daytime in a diurnal manner. Analysis of array data suggested that NDA1 clustered closest to the gene encoding the P-subunit of glycine decarboxylase. Taken together with the diurnal regulation of NDA1 observed here and in other studies, this suggests that NDA1 plays a role in integrating metabolic activities of chloroplasts and mitochondria. NDA2, NDB2 and Aox1a were up-regulated in a coordinated manner under various treatments, potentially forming a complete respiratory chain capable of oxidizing matrix and cytosolic NAD(P)H. NDB1 and NDC1 were down-regulated under the same conditions and may be regarded as housekeeping genes.