Organization and expression of the mitochondrial genome in the Nicotiana sylvestris CMSII mutant.

Previous analyses suggested that the Nicotiana sylvestris CMSII mutant carried a large deletion in its mitochondrial genome. Here, we show by cosmid mapping that the deletion is 60 kb in length and contains several mitochondrial genes or ORFs, including the complex I nad7 gene. However, due to the presence of large duplications in the progenitor mitochondrial genome, the only unique gene that appears to be deleted is nad7. RNA gel blot data confirm the absence of nad7 expression, strongly suggesting that the molecular basis for the CMSII abnormal phenotype, poor growth and male sterility, is the altered complex I structure. The CMSII mitochondrial genome appears to consist essentially of one of two subgenomes resulting from recombination between direct short repeats. In the progenitor mitochondrial genome both recombination products are detected by PCR and, reciprocally, the parental fragments are detected at the substoichiometric level in the mutant. The CMSII mtDNA organization has been maintained through six sexual generations.     

Contribution of Gamma amino butyric acid (GABA) to salt stress responses of Nicotiana sylvestris CMSII mutant and wild type plants

Plants accumulate high levels of Gamma amino butyric acid (GABA) in response to different environmental stresses and GABA metabolism has different functions such as osmotic and pH regulation, bypass of tricarboxylic acid cycle, and C:N balance. The cytoplasmic male sterile (CMS) II mutant of Nicotiana sylvestris has a deletion in the mitochondrial gene nad7 which encodes the NAD7 subunit of complex I which causes increased leaf respiration, impaired photosynthesis, slower growth and increased amino acid levels. In this study we aimed to elucidate the role of GABA and GABA metabolism in different genotypes of the same plant system under salt stress (100mM NaCl) in short (24h) and long (7, 14 and 21 days) terms. We have investigated the differences in leaf fresh and dry weights, relative water content, photosynthetic efficiency (F(v)/F(m)), glutamate dehydrogenase (GDH, EC 1.4.1.4) and glutamate decarboxylase (GAD, EC 4.1.1.15) enzyme activities, GABA content and GAD gene expression profiles. GDH activity showed variations in CMSII and wild type (WT) plants in the first 24h. GAD gene expression profiles were in good agreement with the GAD enzyme activity levels in CMSII and WT plants after 24h. In long-term salinity, GAD activities increased in WT but, decreased in CMSII. GABA accumulation in WT and CMSII plants in short and long term was induced by salt stress. Variations in GDH and GAD activities in relation to GABA levels were discussed and GABA metabolism has been proposed to be involved in better performance of CMSII plants under long term salinity.     

A partially assembled complex I in NAD4-deficient mitochondria of maize

The proton-translocating NADH:ubiquinone oxidoreductase (respiratory complex I) consists of at least 32 subunits in higher plants, nine of which are mitochondrially encoded (NAD 1–7, NAD4L, NAD9). Complex I (CI) has been analyzed from a mitochondrial mutant of maize, NCS2, that carries a deletion for the 3′ end of the nad4 gene. Mitochondria from highly defective, near-homoplasmic mutant plants have only trace amounts of the normal complex I. Instead, a reduced amount of a smaller complex, which also exhibits NADH dehydrogenase activity, is detected on ‘blue-native’ polyacrylamide gels. Subunits of 76 kDa, 40 kDa and 55 kDa, as well as NAD7 and NAD9, have been identified in the subcomplex by their cross-reactivity with heterologous antisera. The corresponding subunits in Neurospora are localized in a ‘peripheral arm’ of CI, which is known to assemble independently of a ‘membrane arm’. The maize NCS2 CI subcomplex is loosely bound to the membrane and is missing several subunits that could be membrane components. Thus, the mutant CI subcomplex may consist of a peripheral arm. A reduction in the steady-state levels of NAD7 and NAD9 in NCS2 mitochondria occurs despite normal rates of biosynthesis and there is a concomitant decrease of the nuclear encoded 76 kDa subunit. The reduction in CI-associated NADH dehydrogenase activity in the nad4 -deficient NCS2 mutant mitochondria is not associated with a compensatory increase in the activities or amounts of the putative ‘exogenous’ NAD(P)H dehydrogenases that are found in plant mitochondria.     

Deletion of the last two exons of the mitochondrialnad7 gene results in lack of the NAD7 polypeptide in aNicotiana sylvestris CMS mutant

InNicotiana sylvestris, two cytoplasmic male sterile (CMS) mutants obtained by protoplast culture show abnormal developmental features of both vegetative and reproductive organs, and mitochondrial gene reorganization following homologous recombination between 65 bp repeated sequences. A mitochondrial region of 16.2 kb deleted from both CMS mutants was found to contain the last two exons of thenad7 gene coding for a subunit of the mitochondrial respiratory chain complex I, which is encoded in the nucleus in fungi and animals but was recently found to be encoded by the mitochondrial genome in wheat. Although theN. sylvestris nad7 gene shows strong homology with its wheat counterpart, it contains only three introns instead of four. Polymerase chain reaction (PCR) experiments indicated that the parental gene organization, including the completenad7 gene, is probably maintained at a substoichiometric level in the CMS mutants, but this proportion is too low to have a significant physiological role, as confirmed by expression studies showing the lack of detectable amounts of the NAD7 polypeptide. Consequently, absence of NAD7 is not lethal to plant cells but a deficiency of complex I could be involved in the abnormal CMS phenotype.     

Deletion of the last two exons of the mitochondrialnad7 gene results in lack of the NAD7 polypeptide in aNicotiana sylvestris CMS mutant

InNicotiana sylvestris, two cytoplasmic male sterile (CMS) mutants obtained by protoplast culture show abnormal developmental features of both vegetative and reproductive organs, and mitochondrial gene reorganization following homologous recombination between 65 bp repeated sequences. A mitochondrial region of 16.2 kb deleted from both CMS mutants was found to contain the last two exons of thenad7 gene coding for a subunit of the mitochondrial respiratory chain complex I, which is encoded in the nucleus in fungi and animals but was recently found to be encoded by the mitochondrial genome in wheat. Although theN. sylvestris nad7 gene shows strong homology with its wheat counterpart, it contains only three introns instead of four. Polymerase chain reaction (PCR) experiments indicated that the parental gene organization, including the completenad7 gene, is probably maintained at a substoichiometric level in the CMS mutants, but this proportion is too low to have a significant physiological role, as confirmed by expression studies showing the lack of detectable amounts of the NAD7 polypeptide. Consequently, absence of NAD7 is not lethal to plant cells but a deficiency of complex I could be involved in the abnormal CMS phenotype.     

L-galactono-1,4-lactone dehydrogenase is required for the accumulation of plant respiratory complex I

Mitochondrial NADH-ubiquinone oxidoreductase (complex I) is the largest enzyme of the oxidative phosphorylation system, with subunits located at the matrix and membrane domains. In plants, holocomplex I is composed of more than 40 subunits, 9 of which are encoded by the mitochondrial genome (NAD subunits). In Nicotiana sylvestris, a minor 800-kDa subcomplex containing subunits of both domains and displaying NADH dehydrogenase activity is detectable. The NMS1 mutant lacking the membrane arm NAD4 subunit and the CMSII mutant lacking the peripheral NAD7 subunit are both devoid of the holoenzyme. In contrast to CMSII, the 800-kDa subcomplex is present in NMS1 mitochondria, indicating that it could represent an assembly intermediate lacking the distal part of the membrane arm. L-galactono-1,4-lactone dehydrogenase (GLDH), the last enzyme in the plant ascorbate biosynthesis pathway, is associated with the 800-kDa subcomplex but not with the holocomplex. To investigate possible relationships between GLDH and complex I assembly, we characterized an Arabidopsis thaliana gldh insertion mutant. Homozygous gldh mutant plants were not viable in the absence of ascorbate supplementation. Analysis of crude membrane extracts by blue native and two-dimensional SDS-PAGE showed that complex I accumulation was strongly prevented in leaves and roots of Atgldh plants, whereas other respiratory complexes were found in normal amounts. Our results demonstrate the role of plant GLDH in both ascorbate biosynthesis and complex I accumulation.     

Mitochondria-driven changes in leaf NAD status exert a crucial influence on the control of nitrate assimilation and the integration of carbon and nitrogen metabolism

The Nicotiana sylvestris mutant, CMS, lacks the mitochondrial gene nad7 and functional complex I, and respires using low-affinity NADH (alternative) mitochondrial dehydrogenases. Here, we show that this adjustment of respiratory pathways is associated with a profound modification of foliar carbon-nitrogen balance. CMS leaves are characterized by abundant amino acids compared to either wild-type plants or CMS in which complex I function has been restored by nuclear transformation with the nad7 cDNA. The metabolite profile of CMS leaves is enriched in amino acids with low carbon/nitrogen and depleted in starch and 2-oxoglutarate. Deficiency in 2-oxoglutarate occurred despite increased citrate and malate and higher capacity of key anaplerotic enzymes, notably the mitochondrial NAD-dependent isocitrate dehydrogenase. The accumulation of nitrogen-rich amino acids was not accompanied by increased expression of enzymes involved in nitrogen assimilation. Partitioning of (15)N-nitrate into soluble amines was enhanced in CMS leaf discs compared to wild-type discs, especially in the dark. Analysis of pyridine nucleotides showed that both NAD and NADH were increased by 2-fold in CMS leaves. The growth retardation of CMS relative to the wild type was highly dependent on photoperiod, but at all photoperiod regimes the link between high contents of amino acids and NADH was observed. Together, the data provide strong evidence that (1) NADH availability is a critical factor in influencing the rate of nitrate assimilation and that (2) NAD status plays a crucial role in coordinating ammonia assimilation with the anaplerotic production of carbon skeletons.     

Evolution of a pseudogene: exclusive survival of a functional mitochondrial nad7 gene supports Haplomitrium as the earliest liverwort lineage and proposes a secondary loss of RNA editing in Marchantiidae

Gene transfer from the mitochondrion into the nucleus is a corollary of the endosymbiont hypothesis. The frequent and independent transfer of genes for mitochondrial ribosomal proteins is well documented with many examples in angiosperms, whereas transfer of genes for components of the respiratory chain is a rarity. A notable exception is the nad7 gene, encoding subunit 7 of complex I, in the liverwort Marchantia polymorpha, which resides as a full-length, intron-carrying and transcribed, but nonspliced pseudogene in the chondriome, whereas its functional counterpart is nuclear encoded. To elucidate the patterns of pseudogene degeneration, we have investigated the mitochondrial nad7 locus in 12 other liverworts of broad phylogenetic distribution. We find that the mitochondrial nad7 gene is nonfunctional in 11 of them. However, the modes of pseudogene degeneration vary: whereas point mutations, accompanied by single-nucleotide indels, predominantly introduce stop codons into the reading frame in marchantiid liverworts, larger indels introduce frameshifts in the simple thalloid and leafy jungermanniid taxa. Most notably, however, the mitochondrial nad7 reading frame appears to be intact in the isolated liverwort genus Haplomitrium. Its functional expression is shown by cDNA analysis identifying typical RNA-editing events to reconstitute conserved codon identities and also confirming functional splicing of the 2 liverwort-specific group II introns. We interpret our results 1) to indicate the presence of a functional mitochondrial nad7 gene in the earliest land plants and strongly supporting a basal placement of Haplomitrium among the liverworts, 2) to indicate different modes of pseudogene degeneration and chondriome evolution in the later branching liverwort clades, 3) to suggest a surprisingly long maintenance of a nonfunctional gene in the presumed oldest group of land plants, and 4) to support the model of a secondary loss of RNA-editing activity in marchantiid liverworts.     

Use of mitochondrial electron transport mutants to evaluate the effects of redox state on photosynthesis, stress tolerance and the integration of carbon/nitrogen metabolism

Primary leaf metabolism requires the co-ordinated production and use of carbon skeletons and redox equivalents in several subcellular compartments. The role of the mitochondria in leaf metabolism has long been recognized, but it is only recently that molecular tools and mutants have become available to evaluate cause-and-effect relationships. In particular, analysis of the CMSII mutant of Nicotiana sylvestris, which lacks functional complex I, has provided information on the role of mitochondrial electron transport in leaf function. The essential feature of CMSII is the absence of a major NADH sink, i.e. complex I. This necessitates re-adjustment of whole-cell redox homeostasis, gene expression, and also influences metabolic pathways that use pyridine nucleotides. In air, CMSII is not able to use its photosynthetic capacity as well as the wild type. The mutant shows up-regulation of the leaf antioxidant system, lower leaf contents of reactive oxygen species, and enhanced stress resistance. Lastly, the loss of a major mitochondrial dehydrogenase has important repercussions for the integration of primary carbon and nitrogen metabolism, causing distinct changes in leaf organic acid profiles, and also affecting downstream processes such as the biosynthesis of the spectrum of leaf amino acids.     

Functional molecular aspects of the NADH dehydrogenases of plant mitochondria

There are multiple routes of NAD(P)H oxidation associated with the inner membrane of plant mitochondria. These are the phosphorylating NADH dehydrogenase, otherwise known as Complex I, and at least four other nonphosphorylating NAD(P)H dehydrogenases. Complex I has been isolated from beetroot, broad bean, and potato mitochondria. It has at least 32 polypeptides associated with it, contains FMN as its prosthetic group, and the purified enzyme is sensitive to inhibition by rotenone. In terms of subunit complexity it appears similar to the mammalian and fungal enzymes. Some polypeptides display antigenic similarity to subunits fromNeurospora crassa but little cross-reactivity to antisera raised against some beef heart complex I subunits. Plant complex I contains eight mitochondrial encoded subunits with the remainder being nuclear-encoded. Two of these mitochondrial-encoded subunits, nad7 and nad9, show homology to corresponding nuclear-encoded subunits inNeurospora crassa (49 and 30 kDa, respectively) and beef heart CI (49 and 31 kDa, respectively), suggesting a marked difference between the assembly of CI from plants and the fungal and mammalian enzymes. As well as complex I, plant mitochondria contain several type-II NAD(P)H dehydrogenases which mediate rotenone-insensitive oxidation of cytosolic and matrix NADH. We have isolated three of these dehydrogenases from beetroot mitochondria which are similar to enzymes isolated from potato mitochondria. Two of these enzymes are single polypeptides (32 and 55 kDa) and appear similar to those found in maize mitochondria, which have been localized to the outside of the inner membrane. The third enzyme appears to be a dimer comprised of two identical 43-kDa subunits. It is this enzyme that we believe contributes to rotenone-insensitive oxidation of matrix NADH. In addition to this type-II dehydrogenases, several observations suggest the presence of a smaller form of CI present in plant mitochondria which is insensitive to rotenone inhibition. We propose that this represents the peripheral arm of CI in plant mitochondria and may participate in nonphosphorylating matrix NADH oxidation.