Analysis of a range of catabolic mutants provides evidence that phytanoyl-coenzyme A does not act as a substrate of the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase complex in Arabidopsis during dark-induced senescence.

The process of dark-induced senescence in plants is not fully understood, however, the functional involvement of an electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO), has been demonstrated. Recent studies have revealed that the enzymes isovaleryl-coenzyme A (CoA) dehydrogenase and 2-hydroxyglutarate dehydrogenase act as important electron donors to this complex. In addition both enzymes play a role in the breakdown of cellular carbon storage reserves with isovaleryl-CoA dehydrogenase being involved in degradation of the branched-chain amino acids, phytol, and lysine while 2-hydroxyglutarate dehydrogenase is exclusively involved in lysine degradation. Given that the chlorophyll breakdown intermediate phytanoyl-CoA accumulates dramatically both in knockout mutants of the ETF/ETFQO complex and of isovaleryl-CoA dehydrogenase following growth in extended dark periods we have investigated the direct importance of chlorophyll breakdown for the supply of carbon and electrons during this process. For this purpose we isolated three independent Arabidopsis (Arabidopsis thaliana) knockout mutants of phytanoyl-CoA 2-hydroxylase and grew them under the same extended darkness regime as previously used. Despite the fact that these mutants accumulated phytanoyl-CoA and also 2-hydroxyglutarate they exhibited no morphological changes in comparison to the other mutants previously characterized. These results are consistent with a single entry point of phytol breakdown into the ETF/ETFQO system and furthermore suggest that phytol is not primarily metabolized by this pathway. Furthermore analysis of isovaleryl-CoA dehydrogenase/2-hydroxyglutarate dehydrogenase double mutants generated here suggest that these two enzymes essentially account for the entire electron input via the ETF complex.

Protein degradation - an alternative respiratory substrate for stressed plants.

In cellular circumstances under which carbohydrates are scarce, plants can metabolize proteins and lipids as alternative respiratory substrates. Respiration of protein is less efficient than that of carbohydrate as assessed by the respiratory quotient; however, under certain adverse conditions, it represents an important alternative energy source for the cell. Significant effort has been invested in understanding the regulation of protein degradation in plants. This has included an investigation of how proteins are targeted to the proteosome, and the processes of senescence and autophagy. Here we review these events with particular reference to amino acid catabolism and its role in supporting the tricarboxylic acid cycle and direct electron supply to the ubiquinone pool of the mitochondrial electron transport chain in plants.

Sphingolipid long chain base phosphates can regulate apoptotic-like programmed cell death in plants.

Sphingolipids are ubiquitous components of eukaryotic cells and sphingolipid metabolites, such as the long chain base phosphate (LCB-P), sphingosine 1 phosphate (S1P) and ceramide (Cer) are important regulators of apoptosis in animal cells. This study evaluated the role of LCB-Ps in regulating apoptotic-like programmed cell death (AL-PCD) in plant cells using commercially available S1P as a tool. Arabidopsis cell cultures were exposed to a diverse array of cell death-inducing treatments (including Cer) in the presence of S1P. Rates of AL-PCD and cell survival were recorded using vital stains and morphological markers of AL-PCD. Internal LCB-P levels were altered in suspension cultured cells using inhibitors of sphingosine kinase and changes in rates of death in response to heat stress were evaluated. S1P reduced AL-PCD and promoted cell survival in cells subjected to a range of stresses. Treatments with inhibitors of sphingosine kinase lowered the temperature which induced maximal AL-PCD in cell cultures. The data supports the existence of a sphingolipid rheostat involved in controlling cell fate in Arabidopsis cells and that sphingolipid regulation of cell death may be a shared feature of both animal apoptosis and plant AL-PCD.

Identification of the 2-hydroxyglutarate and isovaleryl-CoA dehydrogenases as alternative electron donors linking lysine catabolism to the electron transport chain of Arabidopsis mitochondria.

The process of dark-induced senescence in plants is relatively poorly understood, but a functional electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) complex, which supports respiration during carbon starvation, has recently been identified. Here, we studied the responses of Arabidopsis thaliana mutants deficient in the expression of isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase to extended darkness and other environmental stresses. Evaluations of the mutant phenotypes following carbon starvation induced by extended darkness identify similarities to those exhibited by mutants of the ETF/ETFQO complex. Metabolic profiling and isotope tracer experimentation revealed that isovaleryl-CoA dehydrogenase is involved in degradation of the branched-chain amino acids, phytol, and Lys, while 2-hydroxyglutarate dehydrogenase is involved exclusively in Lys degradation. These results suggest that isovaleryl-CoA dehydrogenase is the more critical for alternative respiration and that a series of enzymes, including 2-hydroxyglutarate dehydrogenase, plays a role in Lys degradation. Both physiological and metabolic phenotypes of the isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase mutants were not as severe as those observed for mutants of the ETF/ETFQO complex, indicating some functional redundancy of the enzymes within the process. Our results aid in the elucidation of the pathway of plant Lys catabolism and demonstrate that both isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase act as electron donors to the ubiquinol pool via an ETF/ETFQO-mediated route.

EMB2473/MIRO1, an Arabidopsis Miro GTPase, is required for embryogenesis and influences mitochondrial morphology in pollen.

The regulation of mitochondrial biogenesis, subcellular distribution, morphology, and metabolism are essential for all aspects of plant growth and development. However, the molecular mechanisms involved are still unclear. Here, we describe an analysis of the three Arabidopsis thaliana orthologs of the evolutionarily conserved Miro GTPases. Two of the genes, MIRO1 and MIRO2, are transcribed ubiquitously throughout the plant tissues, and their gene products localize to mitochondria via their C-terminal transmembrane domains. While insertional mutations in the MIRO2 gene do not have any visible impact on plant development, an insertional mutation in the MIRO1 gene is lethal during embryogenesis at the zygote to four-terminal-cell embryo stage. It also substantially impairs pollen germination and tube growth. Laser confocal and transmission electron microscopy revealed that the miro1 mutant pollen exhibits abnormally enlarged or tube-like mitochondrial morphology, leading to the disruption of continuous streaming of mitochondria in the growing pollen tube. Our findings suggest that mitochondrial morphology is influenced by MIRO1 and plays a vital role during embryogenesis and pollen tube growth.

Isolation of intact, functional mitochondria from the model plant Arabidopsis thaliana.

The ability to isolate intact, functional mitochondria from plant tissues is a key technique in the study of the genome, proteome, and metabolic function of the plant mitochondrion. Traditionally, mitochondrial plant researchers have turned to specific plant systems and organs (such as potato tubers and pea shoots) from which mitochondria are readily isolated in large quantities. However, increasingly, research is focused on a small number of model species, and there is a need to adapt existing protocols to allow the isolation of mitochondria from these model species. Arguably, the most important of these is Arabidopsis thaliana, for which a formidable array of genetic resources is available. However, because of its relatively small size and the absence of large heterotrophic organs, Arabidopsis is a challenging plant from which to isolate mitochondria. Here, we present two methods for isolating mitochondria from Arabidopsis, either from heterotrophic cell suspension cultures or from hydroponic seedling cultures. We also present details of commonly used assays to assess the physical and functional integrity of the isolated organelles.

The mitochondrial electron transfer flavoprotein complex is essential for survival of Arabidopsis in extended darkness.

In mammals, the electron transfer flavoprotein (ETF) is a heterodimeric protein composed of two subunits, alpha and beta, that is responsible for the oxidation of at least nine mitochondrial matrix flavoprotein dehydrogenases. Electrons accepted by ETF are further transferred to the main respiratory chain via the ETF ubiquinone oxide reductase (ETFQO). Sequence analysis of the unique Arabidopsis homologues of two subunits of ETF revealed their high similarity to both subunits of the mammalian ETF. Yeast two-hybrid experiments showed that the Arabidopsis ETFalpha and ETFbeta can form a heteromeric protein. Isolation and characterization of two independent T-DNA insertional Arabidopsis mutants of the ETFbeta gene revealed accelerated senescence and early death compared to wild-type during extended darkness. Furthermore in contrast to wild-type, the etfb mutants demonstrated a significant accumulation of several amino acids, isovaleryl CoA and phytanoyl CoA during dark-induced carbohydrate deprivation. These phenotypic characteristics of etfb mutants are broadly similar to those that we observed previously in Arabidopsis etfqo mutants, suggesting functional association between ETF and ETFQO in Arabidopsis, and confirming the essential roles of the ETF/ETFQO electron transfer complex in the catabolism of leucine and involvement in the chlorophyll degradation pathway activated during dark-induced carbohydrate deprivation.

Histochemical staining and quantification of plant mitochondrial respiratory chain complexes using blue-native polyacrylamide gel electrophoresis.

Our knowledge of the respiratory chain and associated defects depends on the study of the multisubunit protein complexes in the inner mitochondrial membrane. Functional analysis of the plant mitochondrial respiratory chain has been successfully achieved by a combination of blue-native polyacrylamide gel electrophoresis (BN-PAGE) for separation of the protein complexes, and in-gel histochemical staining of the enzyme activities. We have optimized this powerful technique by determining linear ranges of amount of protein and enzyme activity for each respiratory complex. Time courses of the in-gel enzyme activities were also performed to determine optimal reaction times. Using the in-gel activity staining method we have previously shown decreased activity of complex V (F(1)F(0)-ATPase) in male-sterile sunflowers (Sabar et al., 2003). Here we have identified unique supercomplexes comprising complex IV (cytochrome c oxidase) in sunflower mitochondria. This method therefore represents a reliable tool for the diagnosis of respiratory dysfunction. In addition, the wider application of BN-PAGE in combination with enzyme activity staining is discussed.

The critical role of Arabidopsis electron-transfer flavoprotein:ubiquinone oxidoreductase during dark-induced starvation.

In mammals, electron-transfer flavoprotein:ubiquinone oxidoreductase (ETFQO) and electron-transfer flavoprotein (ETF) are functionally associated, and ETF accepts electrons from at least nine mitochondrial matrix flavoprotein dehydrogenases and transfers them to ubiquinone in the inner mitochondrial membrane. In addition, the mammalian ETF/ETFQO system plays a key role in beta-oxidation of fatty acids and catabolism of amino acids and choline. By contrast, nothing is known of the function of ETF and ETFQO in plants. Sequence analysis of the unique Arabidopsis thaliana homologue of ETFQO revealed high similarity to the mammalian ETFQO protein. Moreover, green fluorescent protein cellular localization experiments suggested a mitochondrial location for this protein. RNA gel blot analysis revealed that Arabidopsis ETFQO transcripts accumulated in long-term dark-treated leaves. Analysis of three independent insertional mutants of Arabidopsis ETFQO revealed a dramatic reduction in their ability to withstand extended darkness, resulting in senescence and death within 10 d after transfer, whereas wild-type plants remained viable for at least 15 d. Metabolite profiling of dark-treated leaves of the wild type and mutants revealed a dramatic decline in sugar levels. In contrast with the wild type, the mutants demonstrated a significant accumulation of several amino acids, an intermediate of Leu catabolism, and, strikingly, high-level accumulation of phytanoyl-CoA. These data demonstrate the involvement of a mitochondrial protein, ETFQO, in the catabolism of Leu and potentially of other amino acids in higher plants and also imply a novel role for this protein in the chlorophyll degradation pathway activated during dark-induced senescence and sugar starvation.

A Rab-E GTPase mutant acts downstream of the Rab-D subclass in biosynthetic membrane traffic to the plasma membrane in tobacco leaf epidermis.

The function of the Rab-E subclass of plant Rab GTPases in membrane traffic was investigated using a dominant-inhibitory mutant (RAB-E1(d)[NI]) of Arabidopsis thaliana RAB-E1(d) and in vivo imaging approaches that have been used to characterize similar mutants in the plant Rab-D2 and Rab-F2 subclasses. RAB-E1(d)[NI] inhibited the transport of a secreted green fluorescent protein marker, secGFP, but in contrast with dominant-inhibitory RAB-D2 or RAB-F2 mutants, it did not affect the transport of Golgi or vacuolar markers. Quantitative imaging revealed that RAB-E1(d)[NI] caused less intracellular secGFP accumulation than RAB-D2(a)[NI], a dominant-inhibitory mutant of a member of the Arabidopsis Rab-D2 subclass. Furthermore, whereas RAB-D2(a)[NI] caused secGFP to accumulate exclusively in the endoplasmic reticulum, RAB-E1(d)[NI] caused secGFP to accumulate additionally in the Golgi apparatus and a prevacuolar compartment that could be labeled by FM4-64 and yellow fluorescent protein (YFP)-tagged Arabidopsis RAB-F2(b). Using the vacuolar protease inhibitor E64-d, it was shown that some secGFP was transported to the vacuole in control cells and in the presence of RAB-E1(d)[NI]. Consistent with the hypothesis that secGFP carries a weak vacuolar-sorting determinant, it was shown that a secreted form of DsRed reaches the apoplast without appearing in the prevacuolar compartment. When fused to RAB-E1(d), YFP was targeted specifically to the Golgi via a saturable nucleotide- and prenylation-dependent mechanism but was never observed on the prevacuolar compartment. We propose that RAB-E1(d)[NI] inhibits the secretory pathway at or after the Golgi, causing an accumulation of secGFP in the upstream compartments and an increase in the quantity of secGFP that enters the vacuolar pathway.

Coordination of nuclear and mitochondrial genome expression during mitochondrial biogenesis in Arabidopsis.

Mitochondrial biogenesis and function require the regulated and coordinated expression of nuclear and mitochondrial genomes throughout plant development and in response to cellular and environmental signals. To investigate the levels at which the expression of nuclear and mitochondrially encoded proteins is coordinated, we established an Arabidopsis thaliana cell culture system to modulate mitochondrial biogenesis in response to sugar starvation and refeeding. Sucrose deprivation led to structural changes in mitochondria, a decrease in mitochondrial volume, and a reduction in the rate of cellular respiration. All these changes could be reversed by the readdition of sucrose. Analysis of the relative mRNA transcript abundance of genes encoding nuclear and mitochondrially encoded proteins revealed that there was no coordination of expression of the two genomes at the transcript level. An analysis of changes in abundance and assembly of nuclear-encoded and mitochondrially encoded subunits of complexes I to V of the mitochondrial inner membrane in organello protein synthesis and competence for protein import by isolated mitochondria suggested that coordination occurs at the level of protein-complex assembly. These results further suggest that expression of the mitochondrial genome is insensitive to the stress imposed by sugar starvation and that mitochondrial biogenesis is regulated by changes in nuclear gene expression and coordinated at the posttranslational level.

Ceramides induce programmed cell death in Arabidopsis cells in a calcium-dependent manner.

While the role of C2-ceramide in the induction of programmed cell death (PCD) in animal systems has been well documented, little is known of its role in plant cells. Here we show that C2-ceramide induces PCD in Arabidopsis suspension cultures, which is preceded by the generation of a calcium transient and an increase in reactive oxygen species (ROS). Inhibition of the calcium transient prevented cell death, whereas inhibition of ROS had no effect on cell survival. These observations suggest that calcium signalling plays a role in ceramide-induced PCD but is independent of the generation of ROS.

Comparative transcriptome analysis reveals significant differences in gene expression and signalling pathways between developmental and dark/starvation-induced senescence in Arabidopsis.

An analysis of changes in global gene expression patterns during developmental leaf senescence in Arabidopsis has identified more than 800 genes that show a reproducible increase in transcript abundance. This extensive change illustrates the dramatic alterations in cell metabolism that underpin the developmental transition from a photosynthetically active leaf to a senescing organ which functions as a source of mobilizable nutrients. Comparison of changes in gene expression patterns during natural leaf senescence with those identified, when senescence is artificially induced in leaves induced to senesce by darkness or during sucrose starvation-induced senescence in cell suspension cultures, has shown not only similarities but also considerable differences. The data suggest that alternative pathways for essential metabolic processes such as nitrogen mobilization are used in different senescent systems. Gene expression patterns in the senescent cell suspension cultures are more similar to those for dark-induced senescence and this may be a consequence of sugar starvation in both tissues. Gene expression analysis in senescing leaves of plant lines defective in signalling pathways involving salicylic acid (SA), jasmonic acid (JA) and ethylene has shown that these three pathways are all required for expression of many genes during developmental senescence. The JA/ethylene pathways also appear to operate in regulating gene expression in dark-induced and cell suspension senescence whereas the SA pathway is not involved. The importance of the SA pathway in the senescence process is illustrated by the discovery that developmental leaf senescence, but not dark-induced senescence, is delayed in plants defective in the SA pathway.

A proteomic analysis of plant programmed cell death.

Programmed cell death (PCD) is an active cellular suicide that occurs in animals and plants throughout development and in response to both abiotic and biotic stresses. In contrast to animals, little is known about the molecular machinery that regulates plant PCD. We have previously identified transcriptomic changes associated with heat- and senescence-induced PCD in an Arabidopsis cell suspension culture [Plant J. 30 (2002) 431]. However, since plant PCD is also likely to involve elements that are regulated post-transcriptionally, we have undertaken a proteomic analysis in the Arabidopsis system. We identified 11 proteins that increased in abundance relative to total protein in both treatments despite extensive degradation of other proteins. We argue that some of these proteins are maintained during PCD and may therefore have specific functions in the PCD pathway. The increased abundance of several antioxidant proteins as well as a measured increase in free Fe2+ content of the cells indicates an oxidative stress in this system. Several mitochondrial proteins were identified, confirming the importance of this organelle during PCD. We also identified an extracellular glycoprotein that may function in the transmission of a 'death signal' from cell to cell. Putative roles for the identified proteins are presented.

Enzymes of glycolysis are functionally associated with the mitochondrion in Arabidopsis cells.

Mitochondria fulfill a wide range of metabolic functions in addition to the synthesis of ATP and contain a diverse array of proteins to perform these functions. Here, we present the unexpected discovery of the presence of the enzymes of glycolysis in a mitochondrial fraction of Arabidopsis cells. Proteomic analyses of this mitochondrial fraction revealed the presence of 7 of the 10 enzymes that constitute the glycolytic pathway. Four of these enzymes (glyceraldehyde-3-P dehydrogenase, aldolase, phosphoglycerate mutase, and enolase) were also identified in an intermembrane space/outer mitochondrial membrane fraction. Enzyme activity assays confirmed that the entire glycolytic pathway was present in preparations of isolated Arabidopsis mitochondria, and the sensitivity of these activities to protease treatments indicated that the glycolytic enzymes are present on the outside of the mitochondrion. The association of glycolytic enzymes with mitochondria was confirmed in vivo by the expression of enolase- and aldolase-yellow fluorescent protein fusions in Arabidopsis protoplasts. The yellow fluorescent protein fluorescence signal showed that these two fusion proteins are present throughout the cytosol but are also concentrated in punctate regions that colocalized with the mitochondrion-specific probe Mitotracker Red. Furthermore, when supplied with appropriate cofactors, isolated, intact mitochondria were capable of the metabolism of (13)C-glucose to (13)C-labeled intermediates of the trichloroacetic acid cycle, suggesting that the complete glycolytic sequence is present and active in this subcellular fraction. On the basis of these data, we propose that the entire glycolytic pathway is associated with plant mitochondria by attachment to the cytosolic face of the outer mitochondrial membrane and that this microcompartmentation of glycolysis allows pyruvate to be provided directly to the mitochondrion, where it is used as a respiratory substrate.

The intermembrane space of plant mitochondria contains a DNase activity that may be involved in programmed cell death.

The key role for mitochondria in mammalian apoptosis, a form of programmed cell death (PCD), is well established, but a similar role for plant mitochondria is just emerging. In order to unravel the molecular mechanisms linking plant mitochondria to the downstream events of PCD, we have developed an Arabidopsis cell-free system that can be used to monitor biochemical and morphological changes in isolated nuclei that are associated with PCD. Using this system, two activities that resulted in nuclear DNA degradation could be distinguished, both of which were facilitated by the addition of mitochondria. One activity mediated the generation of 30 kb DNA fragments within 3 h and chromatin condensation within 6 h, when nuclei were incubated with mitochondria alone. The second activity required cytosolic extract in addition to mitochondria and resulted in oligonucleosome-sized DNA cleavage after >12 h. Submitochondrial fractionation and pharmacological studies suggested the presence of an Mg2+-dependent nuclease activity in the intermembrane space, which is responsible for the former in vitro activity. The evolutionary conservation of the role of mitochondria in PCD in animals and plants is discussed.

ORFB is a subunit of F1F(O)-ATP synthase: insight into the basis of cytoplasmic male sterility in sunflower.

ORFB is the product of a gene that is conserved in plant mitochondrial genomes, and which, on the basis of sequence motif and structural similarity, is predicted to be the homologue of yeast and mammalian ATP8, part of the F(O) component of the F1F(O)-ATP synthase. We have shown that, in sunflower, orfB transcripts are edited, increasing the similarity of the predicted protein to ATP8 proteins from non-plant species. Blue-native polyacrylamide gel electrophoresis and peptide sequencing confirm that ORFB localizes to the ATP synthase complex. The predicted amino-terminal 19 amino acids of ORFB are identical to those in the chimeric mitochondrial ORF522 protein, which is associated with cytoplasmic male sterility (CMS) in sunflower. Assays comparing respiratory complexes from a male-sterile line expressing ORF522 with those from a male-fertile line show a specific decrease in ATP hydrolysis by the ATP synthase. These observations allow us to propose a mechanism underlying CMS that is associated with the expression of chimeric open reading frames containing part of the orfB gene.

A custom microarray analysis of gene expression during programmed cell death in Arabidopsis thaliana.

Programmed cell death (PCD) is a form of cellular suicide requiring active gene expression, and occurs in both animals and plants. While the cascade of events and the genes that control PCD have been extensively studied in animals, we remain largely ignorant about the similar process in plant cells. Many of the key proteins of animal cell death such as the Bcl-2 family and the caspase family of proteases do not appear to be conserved in plants, suggesting that plants may employ unique mechanisms to execute PCD. To identify genetic elements of PCD in plants, we monitored changes in transcript levels of approximately 100 selected genes during cell death in an Arabidopsis cell suspension culture using a cDNA microarray. PCD was induced in the cell cultures by two independent means (heat treatment or by allowing the cultures to senesce) to allow the distinction to be drawn between changes in gene expression that are related to PCD and those that are specific to a particular treatment. We argue that genes whose expression is altered during PCD induced by two different means may be generally involved in all types of PCD. We show that certain oxidative stress-related genes, including CSD1, CSD3, and GPX, in addition to cysteine proteinases, some transcription factors, and HR-related genes may serve as markers of a core plant cell death programme. Additionally we observe a down-regulation of the mitochondrial adenine nucleotide transporter and suggest that this may be an early event in the execution of plant PCD.