Localization of mitochondria in plant cells by vital staining with rhodamine 123

The positively-charged fluorescent dye rhodamine 123 (r-123) specifically stains mitochondria in living plant protoplasts, suspensionculture cells, and root hairs. This dye functions as a vital stain and permits visualization of the localization, distribution and movement of the mitochondria. Dehydration of root hairs caused mitochondria to aggregate into clumps. Mitochondria were either homogenous or heterogeneous and were frequently seen to accumulate in the perinuclear regions of suspension-culture cells but not in those of protoplasts or root-hair cells. Dinitrophenol and high concentrations of ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid and KCl immediately eliminated fluorescence in r-123-stained mitochondria whereas ionomycin enhanced it. Treatment of seedlings with r-123 resulted in differential brightness of fluorescence in different tissues. Meristematic tissues, such as root and shoot tips, exhibited the brightest fluorescence. The cytotoxicity of r-123 in both germinating seedlings and suspension-culture cells was low. The specificity, sensitivity and low toxicity of r-123 should make it a useful tool in experiments designed to examine agents and conditions which affect the location, the physiological status or the viability of mitochondria.Abbreviations EGTA ethyleneglycol-bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid - DAPI 46-diamidino-2-phenylindole - r-123 rhodamine 123     

Quantitative cytology of the egg and central cell of Plumbago zeylanica and its impact on cytoplasmic inheritance patterns

Summary The egg and central cells of Plumbago zeylanica have an average volume of 543,000 m³ and 2,560,000 m³ respectively, with surface areas of 38,600 m² and 154,000 m². The egg contains an average of 39,900 mitochondria and 730 plastids. The majority of the plastids are perinuclear (> 60%) with less than 40% in lateral areas or near the filiform apparatus. After fertilization, the number of maternal organelles exceeds paternal organelles by a ratio of 11,000 for mitochondria and 154 for plastids. The central cell contains an average of 178,700 mitochondria and 1,840 plastids. After fertilization, these organelles far exceed the number of sperm organelles transmitted, by a ratio of approx. 14,000 for plastids and 1820 for mitochondria. Biparental inheritance of plastids in the embryo is possible, but not favored; the only comparable data in Oenothera and Impatiens reveals that biparental inheritance is possible in up to 124 ratios. Plants lacking biparental plastid inheritance do not contain plastids in the sperm, and thus the presence of even few sperm plastids may result in expression. The number of paternal mitochondria transmitted into the central cell is greater than that transmitted into the egg as the result of preferential fertilization with the mitochondrion-rich dimorphic sperm cell, although the ratio of paternal to maternal mitochondria is 11,000 in the egg and 1820 in the central cell. The similarity in these ratios suggests that there is a critical dosage of mitochondria that is permissible within the zygotic and endospermatic lineages. This may represent either: (1) a maximum permissible value to prevent expression of paternal mitochondrial genome, (2) a minimum ratio required in order to permit recombination of maternal and paternal mitochondrial genomes, or (3) a cytoplasmic genome balance number.Abbreviations mtDNA mitochondrial DNA - S_ua sperm cell unassociated with the vegetative nucleus - S_vn sperm cell physically associated with the vegetative nucleus     

Carnitine-acyltransferase activity of mitochondria from mung-bean hypocotyls

Carnitine-acyltransferase activity assayed with acetyl-CoA, octanoyl-CoA, or palmitoyl-CoA is associated with the mitochondrial but not with the peroxisomes of mung-bean hypocotyls. Using mitochondria as an enzyme source, a half-maximal reaction rate is obtained with a palmitoyl-CoA concentration approximately twice that required with acetyl-CoA. In the presence of a saturating acetyl-CoA concentration the carnitine-acyltransferase activity is not enhanced by palmitoyl-CoA as additional substrate. However, palmitoylcarnitine is formed in addition to acetylcarnitine, and the formation of acetylcarnitine is competitively inhibited by palmitoyl-CoA. It is concluded that the mitochondria of mung-bean hypocotyls possess a carnitine acyltransferase of broad substrate specificity with respect to the chainlength of the acyl-CoA and that the demonstration of a carnitine-palmitoyltransferase activity in plant mitochondria does not indicate the presence of a specific carnitine long-chain acyltransferase.     

β-Oxidation enzymes in the mitochondria of Arum and oilseed rape

-Oxidation enzymes were detected both in the mitochondria and microbodies of Arum maculatum L. spadices and Brassica napus L. seeds. It is apparent that the mitochondrial membrane barrier, which remains intact after sucrose-density-gradient centrifugation, prevents rapid access of acyl-GoA substrates to matrix oxidation tes. Thus intact mitochondria showed little -oxidation enzyme activity. Rupturing of the mitochondrial membrane allowed rapid access of acyl CoAs to matrix sites. Consequently, in ruptured mitochondria, high -oxidation enzyme activities were measured.C. Masterson thanks the Science and Engineering Research Council for the award of a postgraduate student maintenance grant. D.R. Thomas and C. Wood thank their relatives for continuing financial support. The authors also thank West Cumberland Farmers Ltd., Hexham, UK for their gift of oilseed rape seeds.     

cAMP-dependent protein phosphorylation in mitochondria of bovine heart

A study is presented of the cAMP-dependent phosphorylation in bovine heart mitochondria of three proteins of 42, 16 and 6.5 kDa associated to the inner membrane. These proteins are also phosphorylated by the cytosolic cAMP-dependent protein kinase and by the purified catalytic subunit of this enzyme. In the cytosol, proteins of 16 and 6.5 kDa are phosphorylated by the cAMP-dependent kinase. It is possible that cytosolic and mitochondrial cAMP-dependent kinases phosphorylate the same proteins in the two compartments.     

Tri-Calciphor (16,16-dimethyl-15-dehydroprostagalndin B1 trimer)-mediated mitochondrial Ca2+ movements: modulation by phosphate

The trimeric derivative of 16,16-dimethyl-15-dehydroprostaglandin B₁ (termed tri-Calciphor), which protects tissues against ischemic damage, induced Ca²⁺ efflux and swelling in mitochondria in the absence of phosphate, Mg²⁺ and ATP. When glutamate/malate rather than succinate was the substrate, higher tri-Calciphor concentrations were required for the ionophoretic activity. Ca²⁺ efflux and mitochondrial swelling induced by tri-Calciphor were completely inhibited by ATP, phopsphate and Mg²⁺ added together, and partially inhibited with phosphate plus either ATP or Mg²⁺. Between 0 and 7 μM added Ca²⁺ and in the presence of phosphate, ATP and Mg²⁺, tri-Calciphor stimulated the uptake of Ca²⁺ by mitochondria and increased the efficiency of buffering of extramitochondrial Ca²⁺. Thus depending on the assay conditions, two different effects involving Ca²⁺ movements and mitochondria are observed with tri-Calciphor.     

Single anion channels reconstituted from cardiac mitoplasts

Ion channels from sheep cardiac mitoplast (inverted inner mitochondrial membrane vesicle) preparations were incorporated into voltage-clamped planar lipid bilayers. The appearance of anion rather than cation channels could be promoted by exposing the bilayers to osmotic gradients formed by Cl⁻ salts of large, relatively imperment, cations at a pH of 8.8. Two distinct activities were identified. These comprised a multisubstate anion channel of intermediate conductance (∼60 pS in 300vs. 50mm choline Cl, ∼100 pS in symmetric 150mm KCl), and a lower-conductance anion channel (∼25 or ∼50 pS in similar conditions), which only displayed two well-defined substates, at ∼25 and ∼50% of the fully open state. The larger channels were not simple multiples of the lower-conductance channels, but both discriminated poorly, and to a similar extent, between anions and cations (P_Cl ⁻/P_choline ⁺ ∼12, P_Cl ⁻/P_K ⁺∼8). The lower-conductance channel was only minimally selective between different anions (P_{NO 3} ⁻ (1.0)=P_Cl ⁻>P_Br ⁻>P_I ⁻>P_SCN ⁻(0.8)), and its conductance failed to saturate even in high (>1.0 M) activities of KCl. The channels were not obviously voltage dependent, and they were unaffected by 0.5 mM SITS, H₂O₂, propranolol, quinine or amitriptyline, or by 2 mM ATP, or by variations in pH (5.5–8.8). Ca²⁺ and Mg²⁺ did not alter single channel activity, but did modify single current amplitudes in the lower-conductance channel. This effect, together with voltage-dependent substate behavior, is described in the following paper.     

Studies on the behavior of organelles and their nucleoids in the root apical meristem of Arabidopsis thaliana (L.) Col.

The behavior of cell nuclei, mitochondrial nucleoids (mt-nucleoids) and plastid nucleoids (ptnucleoids) was studied in the root apical meristem of Arabidopsis thaliana. Samples were embedded in Technovit 7100 resin, cut into thin sections and stained with 4′-6-diamidino-2-phenylindole for light-microscopic autoradiography and microphotometry. Synthesis of cell nuclear DNA and cell division were both active in the root apical meristem between 0 μm and 300 μm from the central cells. It is estimated that the cells generated in the lower part of the root apical meristem enter the elongation zone after at least four divisions. Throughout the entire meristematic zone, individual cells had mitochondria which contained 1–5 mt-nucleoids. The number of mitochondria increased gradually from 65 to 200 in the meristem of the central cylinder. Therefore, throughout the meristem, individual mitochondria divided either once or twice per mitotic cycle. By contrast, based on the incorporation of [³H]thymidine into organelle nucleoids, syntheses of mitochondrial DNA (mtDNA) and plastid DNA (ptDNA) occurred independently of the mitotic cycle and mainly in a restricted region (i.e., the lower part of the root apical meristem). Fluorimetry, using a videointensified microscope photon-counting system, revealed that the amount of mtDNA per mt-nucleoid in the cells in the lower part of the meristem, where mtDNA synthesis was active, corresponded to more than 1 Mbp. By contrast, in the meristematic cells just below the elongation zone of the root tip, the amount of mtDNA per mt-nucleoid fell to approximately 170 kbp. These findings strongly indicate that the amount of mtDNA per mitochondrion, which has been synthesized in the lower part of the meristem, is gradually reduced as a result of continual mitochondrial divisions during low levels of mtDNA synthesis. This phenomenon would explain why differentiated cells in the elongation zone have mitochondria that contain only extremely small amounts of mtDNA. This work was supported by a Grant-in Aid (T.K.) for Special Research on Priority Areas (Project No. 02242102, Cellular and Molecular Basis for Reproduction Processes in Plants) from the Ministry of Education, Science and Culture of Japan and by a Grant-in Aid (T.K.) for Original and Creative Research Project on Biotechnology from the Research Council, Ministry of Agriculture, Forestry and Fisheries of Japan.     

Ultrastructural changes in the appendix of the Sauromatum guttatum inflorescence during anthesis

Summary The ultrastructure of the epidermal and sub-epidermal cells of the appendix of the Sauromatum guttatum inflorescence reveals developmental changes during anthesis. These changes precede, and probably make possible, heat and odor production. Two days before D-day (the day of heat production and inflorescence-opening) the mitochondria of the epidermis divide; apparent division of the amyloplasts was observed at the same time. The presence of lipid bodies and peroxisomes in the epidermis was clearly evident. On D-day, the epidermis becomes a continuous layer in which the cell walls separating two adjacent cells disappear. At the same time, in the sub-epidermal cells, the mitochondria and the amyloplasts undergo division. The mitochondria become electron-dense, and their DNA is clearly visible. On that day, lipids as well as starch are being depleted. The peroxisomes change in structure every day, from D-2 to D-day. It has also been demonstrated by histochemical techniques that during anthesis the activity of cytochrome c oxidase (3,3-diaminobenzidine as a substrate) decreases whereas the activity of NADH dehydrogenase [tetrazolium salts: nitro-blue tetrazolium chloride (NBT) or neotetrazolium chloride (NT) in the presence of NADH], increases. Oxygen consumption of isolated mitochondria from the D-day appendix was inhibited in the presence of the two tetrazolium salts to a different degree: oxidation of NADH in the presence of NBT was the most sensitive to inhibition, more so than the oxidation of malate and succinate. NT was less effective as an inhibitor in the presence of those three respiratory substrates.     

Cytological evidence of biparental inheritance of plastids and mitochondria inPelargonium

Summary Based on the organelle differences between egg and sperm cells inPelargonium hortorum, the zygote, proembryo, and endosperm were examined under the transmission electron microscope. Plastids and mitochondria in the egg cell are significantly different from those of the sperm cell. Egg plastids are starch-containing and less electron dense. They appear circular, elliptical irregular elongate in sections. Sperm cell plastids are relatively electrondense, mostly cup-shaped or dumbbell and devoid of starch granules. Mitochondria of the egg cell are giant and mostly cup-shaped while sperm mitochondria are smaller and usually circular in section. Double fertilization is completed by 24 h after pollination and the pollen tube can be seen in the degenerated synergid. In the zygote, plastids and mitochondria from male and female gametes can be distinguished by their characteristic differences. Moreover, paternal and maternal organelles appear to be distributed at random in the zygote. Aside from the pollen tube and its released starch granules, there is no enucleated cytoplasmic body in the degenerated synergid. Two days after pollination, the zygote undergoes one transverse division to form a 2-celled proembryo which consists of one larger vacuolated basal cell and one smaller densely cytoplasmic apical cell. Paternal and maternal organelles can be detected in both cells of the proembryo and also in the endosperm at this stage. From these results, it can be concluded that plastids and mitochondria from both male and female gametes have been transmitted into the apical cell of the proembryo and most probably to the following generation.Abbreviations TEM transmission electron microscope - DAPI 4,6-diamidino-2-phenylindole - RFLP restriction fragment length polymorphism