Plant Mitochondria Research Articles

A unique C-terminal domain contributes to the molecular function of Restorer-of-fertility proteins in plant mitochondria.

Restorer-of-fertility (Rf) genes encode pentatricopeptide repeat (PPR) proteins that are targeted to mitochondria where they specifically bind to transcripts that induce cytoplasmic male sterility and repress their expression. In searching for a molecular signature unique to this class of proteins, we found that a majority of known Rf proteins have a distinct domain, which we called RfCTD (Restorer-of-fertility C-terminal domain), and its presence correlates with the ability to induce cleavage of the mitochondrial RNA target. A screen of 219 angiosperm genomes from 123 genera using a sequence profile that can quickly and accurately identify RfCTD sequences revealed considerable variation in RFL/RfCTD gene numbers across flowering plants. We observed that plant genera with bisexual flowers have significantly higher numbers of RFL genes compared to those with unisexual flowers, consistent with a role of these proteins in restoration of male fertility. We show that removing the RfCTD from the RFL protein RNA PROCESSING FACTOR 2-nad6 prevented cleavage of its RNA target, the nad6 transcript, in Arabidopsis thaliana mitochondria. We provide a simple way of identifying putative Rf candidates in genome sequences, new insights into the molecular mode of action of Rf proteins and the evolution of fertility restoration in flowering plants.

Nonspecific Permeability Pore (mPTP) in Plant Mitochondria and Its Role in Cell Death

Nonspecific Permeability Pore (mPTP) in Plant Mitochondria and Its Role in Cell Death

The structural components of the Azotobacter vinelandii iron-only nitrogenase, AnfDKG, form a protein complex within the plant mitochondrial matrix

A long-held goal of synthetic biology has been the transfer of a bacterial nitrogen-fixation pathway into plants to reduce the use of chemical fertiliser on crops such as rice, wheat and maize. There are three classes of bacterial nitrogenase, named after their metal requirements, containing either a MoFe-, VFe- or FeFe-cofactor, that converts N2 gas to ammonia. Relative to the Mo-nitrogenase the Fe-nitrogenase is not as efficient for catalysis but has less complex genetic and metallocluster requirements, features that may be preferable for engineering into crops. Here we report the successful targeting of bacterial Fe-nitrogenase proteins, AnfD, AnfK, AnfG and AnfH, to plant mitochondria. When expressed as a single protein AnfD was mostly insoluble in plant mitochondria, but coexpression of AnfD with AnfK improved its solubility. Using affinity-based purification of mitochondrially expressed AnfK or AnfG we were able to demonstrate a strong interaction of AnfD with AnfK and a weaker interaction of AnfG with AnfDK. This work establishes that the structural components of the Fe-nitrogenase can be engineered into plant mitochondria and form a complex, which will be a requirement for function. This report outlines the first use of Fe-nitrogenase proteins within a plant as a preliminary step towards engineering an alternative nitrogenase into crops.

Interplay of endonucleolytic and exonucleolytic processing in the 3'-end formation of a mitochondrial nad2 RNA precursor in Arabidopsis.

Initiation and termination of plant mitochondrial transcription are poorly controlled steps. Precursor transcripts are thus often longer than necessary, and 3'-end processing as well as control of RNA stability are essential to produce mature mRNAs in plant mitochondria. Plant mitochondrial 3' ends are determined by 3'-to-5' exonucleolytic trimming until the progression of mitochondrial exonucleases along transcripts is stopped by stable RNA structures or RNA binding proteins. In this analysis, we investigated the function of the endonucleolytic mitochondrial stability factor 1 (EMS1) pentatricopeptide repeat (PPR) protein and showed that it is essential for the production and the stabilization of the mature form of the nad2 exons 1-2 precursor transcript, whose 3' end corresponds to the 5' half of the nad2 trans-intron 2. The accumulation of an extended rather than a truncated form of this transcript in ems1 mutant plants suggests that the role of EMS1 in 3' end formation is not strictly limited to blocking the passage of 3'-5' exonucleolytic activity, but that 3' end formation of the nad2 exons 1-2 transcript involves an EMS1-dependent endonucleolytic cleavage. This study demonstrates that the formation of the 3' end of mitochondrial transcripts may involve an interplay of endonucleolytic and exonucleolytic processing mediated by PPR proteins.

Particle bombardment-assisted peptide-mediated gene transfer for highly efficient transient assay

ObjectiveA centrifugation-assisted peptide-mediated gene transfer (CAPT) method was recently developed as an efficient system for gene delivery into plant cells. However, the gene transfer efficiency of CAPT into plant cells was not entirely satisfactory for detecting transient expression of a transgene driven into mitochondria. Here, we report a new gene delivery system using a method called particle bombardment-assisted peptide-mediated gene transfer (PBPT).ResultsWe investigated various parameters of the PBPT method to increase transient gene expression efficiency in Brassica campestris. The optimal conditions for PBPT were a single bombardment with gold particles coated with a DNA‒peptide complex (6 µg of DNA and 2 µg of peptide) at an acceleration pressure of 5 kg/cm2 and a target distance of 12.5 cm. Moreover, bombardment under the optimal conditions successfully transferred the transgene into the cells of other plant species, namely B. juncea and tomato. Thus, we developed a PBPT method for highly efficient delivery of a DNA‒peptide complex into plant mitochondria.

Oxidative and Glycation Damage to Mitochondrial DNA and Plastid DNA during Plant Development.

Oxidative damage to plant proteins, lipids, and DNA caused by reactive oxygen species (ROS) has long been studied. The damaging effects of reactive carbonyl groups (glycation damage) to plant proteins and lipids have also been extensively studied, but only recently has glycation damage to the DNA in plant mitochondria and plastids been reported. Here, we review data on organellar DNA maintenance after damage from ROS and glycation. Our focus is maize, where tissues representing the entire range of leaf development are readily obtained, from slow-growing cells in the basal meristem, containing immature organelles with pristine DNA, to fast-growing leaf cells, containing mature organelles with highly-fragmented DNA. The relative contributions to DNA damage from oxidation and glycation are not known. However, the changing patterns of damage and damage-defense during leaf development indicate tight coordination of responses to oxidation and glycation events. Future efforts should be directed at the mechanism by which this coordination is achieved.

Growth Developmental Defects of Mitochondrial Iron Transporter 1 and 2 Mutants in Arabidopsis in Iron Sufficient Conditions.

Iron is the most abundant micronutrient in plant mitochondria, and it has a crucial role in biochemical reactions involving electron transfer. It has been described in Oryza sativa that Mitochondrial Iron Transporter (MIT) is an essential gene and that knockdown mutant rice plants have a decreased amount of iron in their mitochondria, strongly suggesting that OsMIT is involved in mitochondrial iron uptake. In Arabidopsis thaliana, two genes encode MIT homologues. In this study, we analyzed different AtMIT1 and AtMIT2 mutant alleles, and no phenotypic defects were observed in individual mutant plants grown in normal conditions, confirming that neither AtMIT1 nor AtMIT2 are individually essential. When we generated crosses between the Atmit1 and Atmit2 alleles, we were able to isolate homozygous double mutant plants. Interestingly, homozygous double mutant plants were obtained only when mutant alleles of Atmit2 with the T-DNA insertion in the intron region were used for crossings, and in these cases, a correctly spliced AtMIT2 mRNA was generated, although at a low level. Atmit1 Atmit2 double homozygous mutant plants, knockout for AtMIT1 and knockdown for AtMIT2, were grown and characterized in iron-sufficient conditions. Pleiotropic developmental defects were observed, including abnormal seeds, an increased number of cotyledons, a slow growth rate, pinoid stems, defects in flower structures, and reduced seed set. A RNA-Seq study was performed, and we could identify more than 760 genes differentially expressed in Atmit1 Atmit2. Our results show that Atmit1 Atmit2 double homozygous mutant plants misregulate genes involved in iron transport, coumarin metabolism, hormone metabolism, root development, and stress-related response. The phenotypes observed, such as pinoid stems and fused cotyledons, in Atmit1 Atmit2 double homozygous mutant plants may suggest defects in auxin homeostasis. Unexpectedly, we observed a possible phenomenon of T-DNA suppression in the next generation of Atmit1 Atmit2 double homozygous mutant plants, correlating with increased splicing of the AtMIT2 intron containing the T-DNA and the suppression of the phenotypes observed in the first generation of the double mutant plants. In these plants with a suppressed phenotype, no differences were observed in the oxygen consumption rate of isolated mitochondria; however, the molecular analysis of gene expression markers, AOX1a, UPOX, and MSM1, for mitochondrial and oxidative stress showed that these plants express a degree of mitochondrial perturbation. Finally, we could establish by a targeted proteomic analysis that a protein level of 30% of MIT2, in the absence of MIT1, is enough for normal plant growth under iron-sufficient conditions.

Tetratricopeptide-containing SMALL KERNEL 11 is essential for the assembly of cytochrome c oxidase in maize mitochondria.

Assembly of the functional complexes of the mitochondrial respiratory chain requires sophisticated and efficient regulatory mechanisms. In plants, the subunit composition and assembly factors involved in the biogenesis of cytochrome c oxidase (complex IV) are substantially less defined than in mammals and yeast. In this study, we cloned maize (Zea mays) Small kernel 11 (Smk11) via map-based cloning. Smk11 encodes a mitochondria-localized tetratricopeptide repeat protein. Disruption of Smk11 severely affected the assembly and activity of mitochondrial complex IV, leading to delayed plant growth and seed development. Protein interactions studies revealed that SMK11 might interact with four putative complex IV assembly factors, Inner membrane peptidase 1A (ZmIMP1A), MYB domain protein 3R3 (ZmMYB3R-3), cytochrome c oxidase 23 (ZmCOX23), and mitochondrial ferredoxin 1 (ZmMFDX1), among which ZmMFDX1 might interact with subunits ZmCOX6a and ZmCOX-X1; ZmMYB3R-3 might also interact with ZmCOX6a. The mutation of SMK11 perturbed the normal assembly of these subunits, leading to the inactivation of complex IV. The results of this study revealed that SMK11 serves as an accessory assembly factor required for the normal assembly of subunits into complex IV, which will accelerate the elucidation of the assembly of complex IV in plant mitochondria.

Defining the lipidome of Arabidopsis leaf mitochondria: Specific lipid complement and biosynthesis capacity.

Mitochondria are often considered as the power stations of the cell, playing critical roles in various biological processes such as cellular respiration, photosynthesis, stress responses, and programmed cell death. To maintain the structural and functional integrities of mitochondria, it is crucial to achieve a defined membrane lipid composition between different lipid classes wherein specific proportions of individual lipid species are present. Although mitochondria are capable of self-synthesizing a few lipid classes, many phospholipids are synthesized in the endoplasmic reticulum and transferred to mitochondria via membrane contact sites, as mitochondria are excluded from the vesicular transportation pathway. However, knowledge on the capability of lipid biosynthesis in mitochondria and the precise mechanism of maintaining the homeostasis of mitochondrial lipids is still scarce. Here we describe the lipidome of mitochondria isolated from Arabidopsis (Arabidopsis thaliana) leaves, including the molecular species of glycerolipids, sphingolipids, and sterols, to depict the lipid landscape of mitochondrial membranes. In addition, we define proteins involved in lipid metabolism by proteomic analysis and compare our data with mitochondria from cell cultures since they still serve as model systems. Proteins putatively localized to the membrane contact sites are proposed based on the proteomic results and online databases. Collectively, our results suggest that leaf mitochondria are capable-with the assistance of membrane contact site-localized proteins-of generating several lipid classes including phosphatidylethanolamines, cardiolipins, diacylgalactosylglycerols, and free sterols. We anticipate our work to be a foundation to further investigate the functional roles of lipids and their involvement in biochemical reactions in plant mitochondria.

Fine Tuning of ROS, Redox and Energy Regulatory Systems Associated with the Functions of Chloroplasts and Mitochondria in Plants under Heat Stress.

Heat stress severely affects plant growth and crop production. It is therefore urgent to uncover the mechanisms underlying heat stress responses of plants and establish the strategies to enhance heat tolerance of crops. The chloroplasts and mitochondria are known to be highly sensitive to heat stress. Heat stress negatively impacts on the electron transport chains, leading to increased production of reactive oxygen species (ROS) that can cause damages on the chloroplasts and mitochondria. Disruptions of photosynthetic and respiratory metabolisms under heat stress also trigger increase in ROS and alterations in redox status in the chloroplasts and mitochondria. However, ROS and altered redox status in these organelles also activate important mechanisms that maintain functions of these organelles under heat stress, which include HSP-dependent pathways, ROS scavenging systems and retrograde signaling. To discuss heat responses associated with energy regulating organelles, we should not neglect the energy regulatory hub involving TARGET OF RAPAMYCIN (TOR) and SNF-RELATED PROTEIN KINASE 1 (SnRK1). Although roles of TOR and SnRK1 in the regulation of heat responses are still unknown, contributions of these proteins to the regulation of the functions of energy producing organelles implicate the possible involvement of this energy regulatory hub in heat acclimation of plants.

Mitochondrial DNA Isolation from Plants.

For most eukaryotes, sequencing and assembly of the mitochondrial DNA (mtDNA) is possible by starting the analysis from total cellular DNA, but the exploration of the mtDNA of plants is more challenging because of the low copy number, limited sequence conservation, and complex structure of the mtDNA. The very large size of the nuclear genome of many plant species and the very high ploidy of the plastidial genome further complicate the analysis, sequencing, and assembly of plant mitochondrial genomes. An enrichment of mtDNA is therefore necessary. For this, plant mitochondria are purified prior to mtDNA extraction and purification. The relative enrichment in mtDNA can be assessed by qPCR, while the absolute enrichment can be deduced from the proportion of NGS reads mapping to each of the three genomes of the plant cell. Here we present methods for mitochondrial purification and mtDNA extraction applied to different plant species and tissues, and compare the mtDNA enrichment obtained with the different procedures.

A Bibliometric Study for Plant RNA Editing Research: Trends and Future Challenges.

RNA editing is a post-transcriptional process that introduces changes in RNA sequences encoded by nuclear, mitochondrial, or plastid genomes. To understand the research progress of plant RNA editing, we comprehensively analyze the articles on plant RNA editing from 2001 to 2022 through bibliometric methods. Nucleic Acids Research, Plant Journal and Plant cell are the journals that deserve attention with their high production, total local citation scores (TLCS), and h-indexes. The USA, China, and Germany are the top three countries with highly productive publications. Ulm University, Cornell University, and Chinese Acad Sci are excellent cooperative institutions with a high level of influence in the field, and KNOOP V and TAKENAKA M are good partnership. Plant RNA editing researches concentrate on the subject categories of Biochemistry & Molecular Biology, Plant Sciences, Genetics & Heredity, etc. Plant mitochondria, genome editing and messenger-RNA may be the research hotspots in the future. The main plant RNA editing research tools are JACUSA, SPRINT, and REDO, and the main databases are REDIdb, PED, and dbRES. At present, the research streams are (1) RNA editing sites; (2) Pentapeptide repeat protein (PPR) involved in RNA editing; (3) RNA editing factors. Overall, this article summarizes the research overview of plant RNA editing until 2022 and provides theoretical implications for its possible future directions.

Horizontal transfer of tRNA genes to mitochondrial plasmids facilitates gene loss from fungal mitochondrial DNA

Fungal and plant mitochondria are known to exchange DNA with retroviral plasmids. Transfer of plasmid DNA to the organellar genome is best known and occurs through wholesale insertion of the plasmid. Less well known is the transfer of organellar DNA to plasmids, in particular tRNA genes. Presently, it is unknown whether fungal plasmids can adopt mitochondrial functions such as tRNA production through horizontal gene transfer. In this paper, we studied the exchange of DNA between fungal linear plasmids and fungal mtDNA, mainly focusing on the basidiomycete family Lyophyllaceae. We report at least six independent transfers of complete tRNA genes to fungal plasmids. Furthermore, we discovered two independent cases of loss of a tRNA gene from a fungal mitochondrial genome following transfer of such a gene to a linear mitochondrial plasmid. We propose that loss of a tRNA gene from mtDNA following its transfer to a plasmid creates a mutualistic dependency of the host mtDNA on the plasmid. We also find that tRNA genes transferred to plasmids encode codons that occur at the lowest frequency in the host mitochondrial genomes, possibly due to a higher number of unused transcripts. We discuss the potential consequences of mtDNA transfer to plasmids for both the host mtDNA and the plasmid.

Mitochondria targeted and immobilized ratiometric NIR fluorescent probe for investigating SO2 phytotoxicity in plant mitochondria

Exposure to excessive SO 2 will induce severe phytotoxicity to plants. Dissecting mechanisms of SO 2 phytotoxicity is vital for preventing adverse effect of SO 2 . But unfortunately, the mechanisms of SO 2 phytotoxicity remain elusive. We speculated that interaction of SO 2 derivatives with mitochondria might be an important mechanism for SO 2 phytotoxicity. To authenticate this speculation, it is urgently need to detect SO 2 derivatives in plant mitochondria when it is suffering from SO 2 phytotoxicity. Herein, we presented a novel ratiometric NIR fluorescent probe for highly sensitive and selective detection of SO 2 derivatives in plant mitochondria. Notably, the probe not only could target into the mitochondria, but also can immobilize in the mitochondria to continue detecting SO 2 derivatives even when the mitochondrial membrane potential was collapsed by high concentration of SO 2 derivatives. Importantly, the probe has been successfully employed for monitoring high concentration of SO 2 derivatives in the plant mitochondria when the plant is suffering from SO 2 phytotoxicity. The results demonstrated that the SO 2 derivatives level in plant mitochondria is strictly associated with SO 2 phytotoxicity. Thus, the interaction of SO 2 derivatives with plant mitochondria could be considered as an important mechanism for SO 2 phytotoxicity. • The first fluorescent probe that can detect SO 2 derivatives in plant mitochondria has been rationally developed. • The probe not only targets into mitochondria, but also retains in mitochondria when membrane potential is collapsed. • The interaction of SO 2 derivatives with mitochondria was firstly proved as a key mechanism for SO 2 phytotoxicity.

A de novo gene originating from the mitochondria controls floral transition in Arabidopsis thaliana.

De novo genes created in the plant mitochondrial genome have frequently been transferred into the nuclear genome via intergenomic gene transfer events. Therefore, plant mitochondria might be a source of de novo genes in the nuclear genome. However, the functions of de novo genes originating from mitochondria and the evolutionary fate remain unclear. Here, we revealed that an Arabidopsis thaliana specific small coding gene derived from the mitochondrial genome regulates floral transition. We previously identified 49 candidate de novo genes that induce abnormal morphological changes on overexpression. We focused on a candidate gene derived from the mitochondrial genome (sORF2146) that encodes 66 amino acids. Comparative genomic analyses indicated that the mitochondrial sORF2146 emerged in the Brassica lineage as a de novo gene. The nuclear sORF2146 emerged following an intergenomic gene transfer event in the A. thaliana after the divergence between Arabidopsis and Capsella. Although the nuclear and mitochondrial sORF2146 sequences are the same in A. thaliana, only the nuclear sORF2146 is transcribed. The nuclear sORF2146 product is localized in mitochondria, which may be associated with the pseudogenization of the mitochondrial sORF2146. To functionally characterize the nuclear sORF2146, we performed a transcriptomic analysis of transgenic plants overexpressing the nuclear sORF2146. Flowering transition-related genes were highly regulated in the transgenic plants. Subsequent phenotypic analyses demonstrated that the overexpression and knockdown of sORF2146 in transgenic plants resulted in delayed and early flowering, respectively. These findings suggest that a lineage-specific de novo gene derived from mitochondria has an important regulatory effect on floral transition.

Comprehensive prediction of plant cytoplasmic and apoplastic effectors underlying Erwinia psidii pathogenicity

AbstractErwinia psidii (Eps) is the causal agent of emerging diseases of eucalypt and guava; however, the mechanisms underlying its pathogenicity are not fully understood. Here, we predicted factors involved in the ability of Eps to cause disease on its host plants. For that, the genomes of four Eps strains exhibiting different virulence on eucalypt were sequenced, and hrp/hrc genes coding for the type III secretion system (T3SS), effectors injected into the plant cell cytoplasm through the T3SS (T3SEs) and their plant subcellular localizations, as well as proteins deployed to the host apoplast, were predicted. It was found that Eps possesses a complete hrp/hrc gene cluster based on comparison with Erwinia amylovora. A total of 18 T3SEs were predicted, 11 of which were shared among all strains, none were exclusive to any strain and seven were absent in at least one strain. No sequence variation among strains was found for five T3SE candidates whereas extensive variation was found for six, suggesting the latter may be determinants of virulence differences. The T3SE candidates are predicted to target the plant cell nucleus, cytoplasm, mitochondrion, chloroplast and peroxisome. The predicted apoplastic effector repertoire common to all four strains was over‐represented in proteins of unknown functions or predicted to possess enzymatic activities, among which the most abundant were oxidoreductases and peptidases. Proteins with lytic transglycosylase activity were predicted in strain‐specific apoplastic effector repertoires. These results provide an important framework for future research aimed at uncovering the factors underlying Eps pathogenicity.

The P-type pentatricopeptide repeat protein DWEORG1 is a non-previously reported rPPR protein of Arabidopsis mitochondria

Gene expression in plant mitochondria is mainly regulated by nuclear-encoded proteins on a post-transcriptional level. Pentatricopeptide repeat (PPR) proteins play a major role by participating in mRNA stability, splicing, RNA editing, and translation initiation. PPR proteins were also shown to be part of the mitochondrial ribosome (rPPR proteins), which may act as regulators of gene expression in plants. In this study, we focus on a mitochondrial-located P-type PPR protein—DWEORG1—from Arabidopsis thaliana. Its abundance in mitochondria is high, and it has a similar expression pattern as rPPR proteins. Mutant dweorg1 plants exhibit a slow-growth phenotype. Using ribosome profiling, a decrease in translation efficiency for cox2, rps4, rpl5, and ccmFN2 was observed in dweorg1 mutants, correlating with a reduced accumulation of the Cox2 protein in these plants. In addition, the mitochondrial rRNA levels are significantly reduced in dweorg1 compared with the wild type. DWEORG1 co-migrates with the ribosomal proteins Rps4 and Rpl16 in sucrose gradients, suggesting an association of DWEORG1 with the mitoribosome. Collectively, this data suggests that DWEORG1 encodes a novel rPPR protein that is needed for the translation of cox2, rps4, rpl5, and ccmFN2 and provides a stabilizing function for mitochondrial ribosomes.

Morphology and permeability transitions in plant mitochondria: Different aspects of the same event?

Plant mitochondria are sensitive organelles affected by changing environmental stressors. Upon heat shock or the presence of reactive oxygen species, plant mitochondria undergo in vivo morphological derangements associated with the extensively characterized opening of the mitochondrial permeability transition pore. Nevertheless, the classic mitochondrial permeability transition is known to be triggered by calcium overload causing mitochondrial swelling and dysfunction. Here we review evidence concerning calcium handling, permeability transition and mitochondrial impairments in plants, supporting the notion that the mitochondrial morphology transition is an in vivo indicator of the permeability transition.

Pentatricopeptide repeat protein MITOCHONDRIAL STABILITY FACTOR 3 ensures mitochondrial RNA stability and embryogenesis.

Gene expression in plant mitochondria is predominantly governed at the post-transcriptional level and relies mostly on nuclear-encoded proteins. However, the protein factors involved and the underlying molecular mechanisms are still not well understood. Here, we report on the function of the MITOCHONDRIAL STABILITY FACTOR 3 (MTSF3) protein, previously named EMBRYO DEFECTIVE 2794 (EMB2794), and show that it is essential for accumulation of the mitochondrial NADH dehydrogenase subunit 2 (nad2) transcript in Arabidopsis (Arabidopsis thaliana) but not for splicing of nad2 intron 2 as previously proposed. The MTSF3 gene encodes a pentatricopeptide repeat protein that localizes in the mitochondrion. An MTSF3 null mutation induces embryonic lethality, but viable mtsf3 mutant plants can be generated through partial complementation with the developmentally regulated ABSCISIC ACID INSENSITIVE3 promoter. Genetic analyses revealed growth retardation in rescued mtsf3 plants owing to the specific destabilization of mature nad2 mRNA and a nad2 precursor transcript bearing exons 3 to 5. Biochemical data demonstrate that MTSF3 protein specifically binds to the 3' terminus of nad2. Destabilization of nad2 mRNA induces a substantial decrease in complex I assembly and activity and overexpression of the alternative respiratory pathway. Our results support a role for MTSF3 protein in protecting two nad2 transcripts from degradation by mitochondrial exoribonucleases by binding to their 3' extremities.

Mitochondrial dysfunction associated with ascorbate synthesis in plants

Mitochondria are the major organelles of energy production; however, active mitochondria can decline their energetic role and show a dysfunctional status. Mitochondrial dysfunction was induced by high non-physiological level of L-galactone-1,4-lactone (L-GalL), the precursor of ascorbate (AsA), in plant mitochondria. The dysfunction induced by L-GalL was associated with the fault in the mitochondrial electron partition and reactive oxygen species (ROS) over-production. Using mitochondria from RNAi-plant lines harbouring silenced L-galactone-1,4-lactone dehydrogenase (L-GalLDH) activity, it was demonstrated that such dysfunction is dependent on this enzyme activity. The capacity of alternative respiration was strongly decreased by L-GalL, probably mediated by redox-inactivation of the alternative oxidase (AOX) enzyme. Although, alternative respiration was shown to be the key factor that helps support AsA synthesis in dysfunctional mitochondria. Experiments with respiratory inhibitors showed that ROS formation and mitochondrial dysfunction were more associated with the decline in the activities of COX (cytochrome oxidase) and particularly AOX than with the lower activities of respiratory complexes I and III. The application of high L-GalL concentrations induced proteomic changes that indicated alterations in proteins related to oxidative stress and energetic status. However, supra-optimal L-GalL concentration was not deleterious for plants. Instead, the L-GalLDH activity could be positive. Indeed, it was found that wild type plants performed better growth than L-GalLDH-RNAi plants in response to high non-physiological L-GalL concentrations.