How do plants make mitochondria?

Plant mitochondria can differ in size, shape, number and protein content across different tissue types and over development. These differences are a result of signaling and regulatory processes that ensure mitochondrial function is tuned in a cell-specific manner to support proper plant growth and development. In the last decade, the processes involved in mitochondrial biogenesis are becoming clearer, including; how dormant seeds transition from empty promitochondria to fully functional mitochondria with extensive cristae structures and various biochemical activities, the regulation of nuclear genes encoding mitochondrial proteins via regulators of the diurnal cycle in plants, the mitochondrial stress response, the targeting of proteins to mitochondria and other organelles and connections between the respiratory chain and protein import complexes. All these findings indicate that mitochondrial function is a part of an integrated cellular network, and communication between mitochondria and other cellular processes extends beyond the known exchange or transport of metabolites. Our current knowledge now needs to be used to gain more insight into the molecular components at various levels of this hierarchical and complex regulatory and communication network, so that mitochondrial function can be predicted and modified in a rational manner.     

Several types of mitochondrial ultrastructure in animal cell mitochondria and their relationship to energy production]

Using the mitochondrial suspension of rabit's hepatocytes the interconnection between the ultrastructural reorganization and the power of mitochondria has been studied. Several steady states of these organelles were revealed corresponding to the rest, norm and to the excitement, all being characterized by definite ultrastructural and power-production parameters. It was shown that these physiological states of mitochondria were common to the intact cells. On the basis of the idea on the discrete physiological states of mitochondria, cases of a so-called "variety" of these methods of approach to the study of the interrelation between the structure and function of mitochondria may be used as well for the analysis of some pathological changes of these organelles.     

Plant mitochondrial RNase P

Molecular investigations in mitochondria of higher plants have to take in account the complicated genomic structure of these organelles and their complex mode of gene expression. Recently tRNA processing activities and particulary RNase P-like activities have been described for mitochondria of mono- and dicot plants. The determined biochemical characteristics of these plant mitochondrial tRNA processing enzymes now allow a comparison to the bacterial prototype from which they evolved. The substrate specifity of the plant mitochondrial RNase P in particular has unique selection parameters distinct from theE. coli RNase P.     

VARIATIONS IN THE STRUCTURE OF MITOCHONDRIA

A characteristic internal structure, consisting of a double-layered outer wall enclosing a matrix-filled space through which pass double-layered membranous folds, would appear to comprise as satisfactory a definition of mitochondria for electron microscopy as their intravital affinity for Janus green affords for light microscopy. Relying for identification upon this characteristic internal structure, mitochondria appear to be pleomorphic structures which vary in size, shape, complexity, and density. They are labile also in that their number may increase or decrease under controlled conditions. The possibility therefore exists that these organelles are constantly being formed and destroyed, perhaps by their participation in metabolic processes. The problem of the origin of mitochondria is in an unsatisfactory state. New organelles unquestionably are formed in particular physiological states. The possibility that new bodies are produced by fission of ones already present does not seem adequate. On the other hand, the possible fabrication of new mitochondria out of intracellular membranes, although an attractive hypothesis, has not been adequately substantiated.     

Dynamic proteomic analysis reveals diurnal homeostasis of key pathways in rice leaves

Diurnal physiological acclimation regulated by a circadian system is an advantage for plant fitness. The circadian system is composed of a signal input, the clock and output pathways. Understanding the regulation mechanism of the output pathways remains a major challenge. Diurnal proteomic change reflects the state of circadian organization. We found the content of glucose, fructose, sucrose and starch diurnally changed in leaves of rice seedlings grown under a 12-h light/12-h dark condition with constant temperature. Dynamic proteomics analysis revealed 140 protein spots with diurnally changed levels at six times of the light/dark cycle; 132 spots were identified by MS, and 119 spots were of a single protein each with functional annotation. These proteins are involved in regulation of carbohydrate flow, redox, protein folding, nitrogen and protein metabolism, energy conversion, photorespiration and photosynthesis. Of these proteins, 81.5% were upregulated during the light phase, overlappingly, 41.2% showed behavior of circadian anticipation to dawn. Pattern analysis showed that the diurnal regulation involved pathways of allocation of carbohydrates between temporary reserves and consumption, maintenance of redox homeostasis, diurnal protein reassembly and nitrogen assimilation. These pathways reflect biochemical phenotypes of the circadian change linking the oscillator and circadian outputs.     

Differentiation of cellular and biochemical features of the single-cell C4 syndrome during leaf development in Bienertia cycloptera (Chenopodiaceae)

The terrestrial plant Bienertia cycloptera has been shown to accomplish C(4) photosynthesis within individual chlorenchyma cells by spatially separating the phases of carbon assimilation into distinct peripheral and central compartments. In this study, anatomical, physiological, and biochemical techniques were used to determine how this unique compartmentation develops. Western blots show ribulose-1,5-bisphosphate carboxylase (Rubisco) (chloroplastic) is present in the youngest leaves and increases during development, while levels of C(4) enzymes-pyruvate,Pi dikinase (chloroplastic), phosphoenolpyruvate carboxylase (PEPC) (cytosol), and NAD-malic enzyme (mitochondrial)-increase later in development. Immunolocalization confirmed this for Rubisco and PEPC. The youngest chlorenchyma cells have a central nucleus surrounded by monomorphic granal chloroplasts containing Rubisco. Later stages show progressive development of a central cytoplasmic compartment enriched with chloroplasts and mitochondria and of a peripheral cytoplasm with chloroplasts. A complex reticulum of connections between the compartments also developed and was characterized. δ(13)C isotope analyses show mature leaves have distinct C(4)-type isotope composition, while the composition in younger leaves is "C(4)-like." Based on the results, this form of single-cell C(4) photosynthesis develops from a common pool of organelles through partitioning to separate compartments, and the development of biochemically and ultrastructurally dimorphic chloroplasts.     

Optimization of photosynthesis by multiple metabolic pathways involving interorganelle interactions: resource sharing and ROS maintenance as the bases

The bioenergetic processes of photosynthesis and respiration are mutually beneficial. Their interaction extends to photorespiration, which is linked to optimize photosynthesis. The interplay of these three pathways is facilitated by two major phenomena: sharing of energy/metabolite resources and maintenance of optimal levels of reactive oxygen species (ROS). The resource sharing among different compartments of plant cells is based on the production/utilization of reducing equivalents (NADPH, NADH) and ATP as well as on the metabolite exchange. The responsibility of generating the cellular requirements of ATP and NAD(P)H is mostly by the chloroplasts and mitochondria. In turn, besides the chloroplasts, the mitochondria, cytosol and peroxisomes are common sinks for reduced equivalents. Transporters located in membranes ensure the coordinated movement of metabolites across the cellular compartments. The present review emphasizes the beneficial interactions among photosynthesis, dark respiration and photorespiration, in relation to metabolism of C, N and S. Since the bioenergetic reactions tend to generate ROS, the cells modulate chloroplast and mitochondrial reactions, so as to ensure that the ROS levels do not rise to toxic levels. The patterns of minimization of ROS production and scavenging of excess ROS in intracellular compartments are highlighted. Some of the emerging developments are pointed out, such as model plants, orientation/movement of organelles and metabolomics.     

Structural and biochemical dissection of photorespiration in hybrids differing in genome constitution between Diplotaxis tenuifolia (C3-C4) and radish (C3)

We compared the structural, biochemical, and physiological characteristics involved in photorespiration of intergeneric hybrids differing in genome constitution (DtDtR, DtDtRR, and DtRR) between the C(3)-C(4) intermediate species Diplotaxis tenuifolia (DtDt) and the C(3) species radish (Raphanus sativus; RR). The bundle sheath (BS) cells in D. tenuifolia included many centripetally located chloroplasts and mitochondria, but those of radish had only a few chloroplasts and mitochondria. In the hybrids, the numbers of chloroplasts and mitochondria, the ratio of centripetally located organelles to total organelles, and the mitochondrial size in the BS cells increased with an increase in the constitution ratio of the Dt:R genome. The P-protein of glycine decarboxylase (GDC) was confined to the BS mitochondria in D. tenuifolia, whereas in radish, it accumulated more densely in the mesophyll than in the BS mitochondria. In the hybrids, more intense accumulation of GDC in the BS relative to the mesophyll mitochondria occurred with an increase in the Dt:R ratio. These structural and biochemical features in the hybrids were reflected in the gas exchange characteristics of leaves, such as the CO(2) compensation point. Our data indicate that the leaf structure, the intercellular pattern of GDC expression, and the gas exchange characteristics of C(3)-C(4) intermediate photosynthesis are inherited in the hybrids depending on the constitution ratio of the parent genomes. Our findings also demonstrate that the apparent reduced photorespiration in C(3)-C(4) intermediate plants is mainly due to the structural differentiation of mitochondria and chloroplasts in the BS cells combined with the BS-dominant expression of GDC.     

Mitochondrial respiration of the photosynthesizing cell

Current notions on respiration of photosynthesizing cells are reviewed. Over the past three decades, the modern methods based on isotope techniques and reverse and molecular genetics provided convincing evidence that mitochondrial respiration is functional in the light and contributes to the creation of optimal conditions for photosynthesis and for protection of cells from photodegradation. Novel data are presented on the substrates that are used for respiration in the light. Individual respiration steps are considered in the context of their possible role in photosynthesizing cells. The mechanisms and carriers mediating the export of reducing equivalents from chloroplasts for their subsequent oxidation in the mitochondrial electron-transport chain are discussed. The regulation of nonphosphorylating (unrelated to energy generation) electron transport pathways mediated by alternative oxidase and alternative type II NADPH-dehydrogenases, as well as the role of uncoupling proteins in plant mitochondria, are analyzed. These components were shown to play a significant role in NAD(P)H oxidation for maintaining the redox balance in mitochondria and whole green cells. A generalized scheme of biochemical interactions between organelles—chloroplasts, mitochondria, and peroxisomes—is presented. The directions for future research are outlined.     

Ultrastructure and function of mitochondria in gametocytic stage of Plasmodium falciparum

Morphological properties of the mitochondrial organelles in the asexual and sexual gametocytic stages of Plasmodium falciparum have been analyzed and found to be markedly different. From in vitro cultures of both stages in human erythrocytes, it has been demonstrated that the asexual stages contained a defined double-membrane organelle having a few tubular-like cristae. The numbers of mitochondria in the gametocytes were found to be approximately 6 organelles per parasite, and they showed a greater density of the cristae than that of the asexual stage parasite. The organelles of the gametocytes were successfully purified by differential centrifugation following Percoll density gradient separation with the results of approximately 7% yields and approximately 5 folds. The gametocytic organelles contained much more activities of mitochondrial electron transporting enzymes (i.e., cytochrome c reductase, cytochrome c oxidase) than the asexual stage organelles. Mitochondrial function as measured by oxygen consumption were found to be different between these two stages organelles. Their rates of oxygen consumption were relatively low, as compared to those of human leukocyte and mouse liver mitochondria. In contrast to the coupled mammalian mitochondria, the gametocytic organelles were in the uncoupling state between oxidation and phosphorylation reactions during their respiration. However, they were sensitive to inhibitors of the electron transport system, e.g., antimycin A, cyanide. Our results suggest that the mitochondria of the gametocytic stages are metabolically active and still underdeveloped, although their inner membranes are extensively folded. The biochemical significance of the unique structure of the mitochondria in these developing stages in host erythrocytes remains to be elucidated.     

Chloroplast and Mitochondrial Mechanisms for Protection Against Oxygen Toxicity

As a consequence of their oxygen rich environment, organelles of photosynthetic tissues are exposed to large fluxes of oxyradicals and reactive oxygen species. Superoxide, hydrogen peroxide, hydroxyl radical and singlet oxygen are all potential by-products of respiratory and photosynthetic systems. Strong reduc-tants found in mitochondria and chloroplasts along with a steady flux of photosynthetically generated oxygen enhance the potential for oxyradical production. Unless ncturalized by scavenger substrates or enzymes, these reactive intermediates pose a lethal threat.

The presence of superoxide dismutases, cdtalases. various peroxidases and scavenger substrates are all means of defences available to protect organelles. A balance between oxyradical production and neutralization should exist. Perturbations in generation or in sequestration caused by environmental or nutritional factors might profoundly alter the steady state level of oxyintermediates.     

Block of mitochondrial apoptotic pathways in lizard ovarian follicle cells as an adaptation to their nurse function

Pyriforms are ovarian follicle nurse cells that undergo apoptosis at the end of previtellogenesis and are completely eliminated by the epithelium. This event is accompanied by the active transfer of organelles and macromolecules to the oocyte via an intercellular bridge. Since it would be a nonsense for damaged mitochondria to reach the oocyte, we have postulated that pyriform cells have adapted their apoptotic machinery to prevent mitochondrial degradation. To verify this hypothesis, we have studied mitochondrial morphology and functionality during follicle cell regression. Cytological and biochemical evidence indicates that mitochondria in pyriforms maintain their size, organization and membrane potential. This clearly indicates that they are not involved in apoptosis signalling/progression. This block would favour both the oocyte, by increasing the pool of organelles available from follicle cells, and also the regressing pyriforms, by maintaining the energy resources required for completion of their nurse function. The block is probably attributable to an over-expression of Bcl-2 and might be carried out by sequestering cytochrome c inside the organelles. As demonstrated by in vitro experiments, the mitochondrial apoptosis pathway can be activated by stress induction, such as serum deprivation, but not following physiological pro-apoptotic signalling, such as treatment with gonadotrophin-releasing hormone. These studies were supported by a grant from the MIUR (PRIN project: Molecular responses of embryonic, differentiated and tumoral cells exposed to cadmium intoxication).     

Calcium regulation in endosymbiotic organelles of plants

In plant cells calcium-dependent signaling pathways are involved in a large array of biological processes in response to hormones, biotic/abiotic stress signals and a variety of developmental cues. This is generally achieved through binding of calcium to diverse calcium-sensing proteins, which subsequently control downstream events by activating or inhibiting biochemical reactions. Regulation by calcium is considered as a eukaryotic trait and has not been described for prokaryotes. Nevertheless, there is increasing evidence indicating that organelles of prokaryotic origin, such as chloroplasts and mitochondria, are integrated into the calcium-signaling network of the cell. An important transducer of calcium in these organelles appears to be calmodulin. In this review we want to give an overview over present data showing that endosymbiotic organelles harbour calcium-dependent biological processes with a focus on calmodulin-regulation.Key words: mitochondria, chloroplasts, calcium, calmodulin, EF-hand proteins     

Subcellular immunocytochemical analysis detects the highest concentrations of glutathione in mitochondria and not in plastids

The tripeptide glutathione is a major antioxidant and redox buffer with multiple roles in plant metabolism. Glutathione biosynthesis is restricted to the cytosol and the plastids and the product is distributed to the various organelles by unknown mechanisms. In the present study immunogold cytochemistry based on anti-glutathione antisera and transmission electron microscopy was used to determine the relative concentration of glutathione in different organelles of Arabidopsis thaliana leaf and root cells. Glutathione-specific labelling was detected in all cellular compartments except the apoplast and the vacuole. The highest glutathione content was surprisingly not found in plastids, which have been described before as a major site of glutathione accumulation, but in mitochondria which lack the capacity for glutathione biosynthesis. Mitochondria of both leaf and root cells contained 7-fold and 4-fold, respectively, higher glutathione levels than plastids while the density of glutathione labelling in the cytosol, nuclei, and peroxisomes was intermediate. The accuracy of the glutathione labelling is supported by two observations. First, pre-adsorption of the anti-glutathione antisera with glutathione reduced the density of the gold particles in all organelles to background levels. Second, the overall glutathione-labelling density was reduced by about 90% in leaves of the glutathione-deficient Arabidopsis mutant pad2-1 and increased in transgenic plants with enhanced glutathione accumulation. Hence, there was a strong correlation between immunocytochemical and biochemical data of glutathione accumulation. Interestingly, the glutathione labelling of mitochondria in pad2-1 remained very similar to wild-type plants thus suggesting that the high mitochondrial glutathione content is maintained in a situation of permanent glutathione-deficiency at the expense of other glutathione pools. High and constant levels of glutathione in mitochondria appear to be particularly important in cell survival strategies and it is predicted that mitochondria must have highly competitive mitochondrial glutathione uptake systems. The present results underline the suggestion that subcellular glutathione concentrations are not controlled by a global mechanism but are controlled on an individual basis and it is therefore not possible to conclude from global biochemical glutathione analysis on the status of the various organellar pools.