Cellular organisation in meiotic and early post-meiotic rice anthers

We have used fluorescent, confocal laser and transmission electron microscopy (TEM) to examine cellular organisations, including callose (1,3-beta-glucan) behaviour, in meiotic and early post-meiotic rice anthers. These features are critical for pollen formation and provide information to better understand pollen sterility caused by abiotic stress in rice and other monocotyledonous species. Among organelles during meiosis, abundant plastids, mitochondria and nuclei of the anther cells show distinctive features. Chloroplasts in the endothecium store starch and indicate a potential for photosynthetic activity. During meiosis, the middle layer cells are markedly compressed and at the tetrad stage are either vacuolated or filled with degenerating electron-opaque organelles. Viable mitochondria, stained with Rhodamine 123, are seen in the endothecium and tapetum, but the mitochondria in the middle layer are not stained during meiosis. The radial walls of the tapetum are disorganised and degenerating, indicating the formation of a syncytium; pro-orbicules are located at the locular walls at the tetrad stage. Immunohistochemical studies show that the sporogenous cells are entirely enveloped by a thick callosic layer at early meiosis. Cell plate callose was assembled in a plane between the dyad cells. In the tetrads, however, callose formed only at the centre, showing that the tetrad microspores are not enveloped but separated by callose walls. Thick, undulating electron-opaque walls around the tetrads indicate the beginning of exinous microspore wall differentiation.     

Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties

The spectral and metabolic properties of Rhodamine 123, a fluorescent cationic dye used to label mitochondria in living cells, were investigated in suspensions of isolated rat-liver mitochondria. A red shift of Rhodamine 123 absorbance and fluorescence occurred following mitochondrial energization. Fluorescence quenching of as much as 75% also occurred. The red shift and quenching varied linearly with the potassium diffusion potential, but did not respond to delta pH. These energy-linked changes were accompanied by dye uptake into the matrix space. Concentration ratios, in-to-out, approached 4000:1. A large fraction of internalized dye was bound. At concentrations higher than those needed to record these spectral changes, Rhodamine 123 inhibited ADP-stimulated (State 3) respiration of mitochondria (Ki = 12 microM) and ATPase activity of inverted inner membrane vesicles (Ki = 126 microM) and partially purified F1-ATPase (Ki = 177 microM). The smaller Ki for coupled mitochondria was accounted for by energy-dependent Rhodamine 123 uptake into the matrix. Above about 20 nmol/mg protein (10 microM), Rhodamine 123 caused rapid swelling of energized mitochondria. Effects on electron-transfer reactions and coupling were small or negligible even at the highest Rhodamine 123 concentrations employed. delta psi-dependent Rhodamine 123 uptake together with Rhodamine 123 binding account for the intense fluorescent staining of mitochondria in living cells. Inhibition of mitochondria ATPase likely accounts for the cytotoxicity of Rhodamine 123. At concentrations which do not inhibit mitochondrial function, Rhodamine 123 is a sensitive and specific probe of delta psi in isolated mitochondria.     

Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties

The spectral and metabolic properties of Rhodamine 123, a fluorescent cationic dye used to label mitochondria in living cells, were investigated in suspensions of isolated rat-liver mitochondria. A red shift of Rhodamine 123 absorbance and fluorescence occurred following mitochondrial energization. Fluorescence quenching of as much as 75% also occurred. The red shift and quenching varied linearly with the potassium diffusion potential, but did not respond to ΔpH. These energy-linked changes were accompanied by dye uptake into the matrix space. Concentration ratios, in-to-out, approached 4000:1. A large fraction of internalized dye was bound. At concentrations higher than those needed to record these spectral changes, Rhodamine 123 inhibited ADP-stimulated (State 3) respiration of mitochondria (K_i = 12 μM) and ATPase activity of inverted inner membrane vesicles (K_i = 126 μM) and partially purified F₁-ATPase (K_i = 177 μM). The smaller K_i for coupled mitochondria was accounted for by energy-dependent Rhodamine 123 uptake into the matrix. Above about 20 nmol/mg protein (10 μM), Rhodamine 123 caused rapid swelling of energized mitochondria. Effects on electron-transfer reactions and coupling were small or negligible even at the highest Rhodamine 123 concentrations employed. Δψ-dependent Rhodamine 123 uptake together with Rhodamine 123 binding account for the intense fluorescent staining of mitochondria in living cells. Inhibition of mitochondria ATPase likely accounts for the cytotoxicity of Rhodamine 123. At concentrations which do not inhibit mitochondrial function, Rhodamine 123 is a sensitive and specific probe of Δψ in isolated mitochondria.     

Mitochondrial uptake of rhodamine 123 by rabbit articular chondrocytes

Rhodamine 123 was used to stain and analyze by flow cytometry the mitochondria of rabbit articular chondrocytes. Stationary primary cultures and exponentially growing subcultures were compared to enzymatically released chondrocytes from cartilage. The increase in mitochondrial fluorescence, when chondrocytes are transferred from cartilage to culture environment, is suggestive of some change in chondrocyte adaptation and/or differentiation in these conditions.     

Rhodamine 123 as a vital stain for mitochondria of plant cells

Abstract. Rhodamine 123 (Rh 123), a relatively new mitochondrial marker, little used in the study of plant cells, was tested on excized leaves of Elodea canadensis Michx. and on suspension-cultured cells of Ranunculus serbicus Vis. In both preparations, the dye accumulated rapidly and selectively in the mitochondria whose number, morphology and cell distribution could be easily observed. In the presence of Rh 123, cytoplasmic movements could also be perceived and the spatial arrangement of the mitochondria with respect to that of the auto-fluorescent chloroplasts was studied in connection with a normal or altered cytoskeletal framework. The specific uptake of Rh 123 by the organdies seemed to be potential-dependent since it was influenced by cations, ionophores and inhibitors of electron transport. Short exposures to the stain were practically non-toxic, whereas prolonged treatments (6–20 h) provoked specific alterations in structure of the mitochondria. The data reported here indicate that Rh 123 may be an excellent vital stain to study the morphology, function and dynamics of the mitochondria in living plant cells.     

油菜雄性核不育系及其等位可育系小孢子发育过程的比较研究

用Olympus BH2型光学显微镜对甘蓝型油菜单显性核不育系(GMS)及其等位可育系小孢子发育过程进行解剖学观察,发现在正常小孢子发育过程中,绒毡层在小孢子发育的四分体前后开始解体,为小孢子继续发育提供营养,而不育系小孢子的败育在减数分裂前就已经发生,并且不能形成四分体,小孢子逐渐解体,且小孢子解体在绒毡层解体之前发生,最后花药成为干瘪的空壳,导致不育。     

Rhodamine 123 inhibits bioenergetic function in isolated rat liver mitochondria

Rhodamine 123 accumulates in the mitochondria of living cells and exhibits selective anticarcinoma activity. The biochemical basis of toxicity was investigated by testing the effect of the dye on isolated rat liver mitochondria. Much lower concentrations of rhodamine 123 were required to inhibit ADP-stimulated respiration and ATP synthesis in well-coupled energized mitochondria than were required to inhibit uncoupled respiration and uncoupler-stimulated ATP hydrolysis. The amount of rhodamine 123 associated with the mitochondria was several-fold greater under energized as compared to non-energized conditions, which may explain why coupled functions appeared to be more sensitive than uncoupled functions to inhibition at low concentrations of rhodamine 123. It was concluded that the site of rhodamine 123 inhibition is most likely the F0F1 ATPase complex and possibly electron transfer reactions as well.     

A COMPARATIVE LIGHT- AND ELECTRON-MICROSCOPIC STUDY OF MICROSPOROGENESIS IN MALE-FERTILE AND CYTOPLASMIC MALE-STERILE SUNFLOWER (HELIANTHUS ANNUUS)

Cytoplasmic male sterility (CMS) in sunflower anthers is compared with its normal (N) line by using light and electron microscopy. Degeneration and disintegration of CMS tapetum and microspore tetrads occur after meiosis II, resulting in sterility. At the onset of meiosis, the CMS tapetum enlarges radially and shows signs of disorganization of organelles and walls. The developing CMS meiocytes and tetrads of microspores do not show these abnormalities when compared with their N counterparts. The CMS microspore tetrads remain viable until a rudimentary exine forms around each microspore. At this time, the radially enlarged tapetum disintegrates, followed by disintegration of the tetrads. In N-line microsporogenesis, a peripheral, dense tapetum is present at the tetrad stage, and as each locule enlarges, free spaces occur around the tetrads. After a rudimentary exine with associated spines and colpi is formed around each microspore, the callose holding each tetrad together dissolves, freeing the microspores for further development. Eventually the binucleate tapetum becomes plasmodial, persisting until the vacuolate pollen stage.     

Plant mitochondrial electrical potential monitored by fluorescence quenching of rhodamine 123

The suitability of the fluorescent dye rhodamine 123 for qualitative and quantitative determinations of the electrical potential difference (ΔΨ) in isolated pea (Pisum sativum L.) stem mitochondria was evaluated. A fluorescence quenching of rhodamine 123, as a consequence of dye uptake, occurred following mitochondria energization by both external and internal substrates. This quenching was associated to the generation of ΔΨ, because it was completely released by uncouplers and respiratory inhibitors. The conversion of the proton gradient (ΔpH) into ΔΨ, induced by nigericin or a permeant weak acid (phosphate), increased the quenching. The uptake of the probe was accompanied by 40 % of unspecific binding in coupled, but not in uncoupled, mitochondria. Rhodamine 123 quenching varied linearly with a K+-diffusion potential. ADP induced a transient and cyclic change of fluorescence which was associated to ATP synthesis. Consequently, rhodamine 123 did not influence oxygen consumption by mitochondria in both state 4 and 3, thus indicating that, at the concentrations assayed, the probe was not toxic. It is concluded that rhodamine 123, followed by fluorescence quenching, is a suitable probe to study the energetics of isolated plant mitochondria. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of pollen load size and source (self, outcross) on seed and fruit production in highbush blueberry cv. 'Bluecrop' (VACCINIUM CORYMBOSUM; Ericaceae)

Reproductive fitness of a plant is ultimately determined by both number and quality of seed offspring. This is determined by sexual selection of pollen microspores and ovules during pollination and fertilization. These processes may include pollen competition and seed abortion, which reduce the number of microspores and ovules available for final seed production. Thus, even an excess of pollen microspores to ovules does not result in fertile seeds equal to ovule number. We investigated pollen requirements of highbush blueberry (Vaccinium corymbosum cultivar 'Bluecrop') for maximal seed production and how fertile seed number translates into fruit quality, since fruit quality would ultimately determine the dispersal of its offspring. We demonstrate that individual blueberry flowers with a mean of 106 ovules reach their maximum fruit set and mass and minimum time to ripen when 125 outcross pollen tetrads pollinate a flower, compared to 10 or 25. Three hundred tetrads resulted in the increase of fertile seeds, but did not result in a further increase of fruit mass or fruit set, or decrease in time to ripen. We also examined the effect of pure and mixed loads of self and outcross pollen (25 and 125 tetrads), and found no differences in fertile seed number, fruit mass, or percentage fruit set when pollen loads were either 25 self or outcross pollen tetrads, although number of days to ripen was significantly shorter by 8 d with 25 outcross tetrads. When the pollen load of 125 tetrads consisted of self or a 50:50 mixture of self and outcross pollen, fruit mass, days to ripen, and percentage fruit set were not different from loads of 125 outcross pollen. In addition, a pollen load of 25 outcross tetrads resulted in fertile seed number and fruit quality in between that of 25 self, and 125 self, 125 mixed, or 125 outcross tetrads. Large, small, and flat seed types were identified, and only large seeds (length = 1.7 mm) were fertile. These results improve our understanding of pollen load size and source requirements of a crop plant and the limits to pollen transfer when translated to fruit growth.     

The Arabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation/condensation complexes

The Arabidopsis thaliana MALE STERILITY 2 ( MS2 ) gene product is involved in male gametogenesis. The first abnormalities in pollen development of ms2 mutants are seen at the stage in microsporogenesis when microspores are released from tetrads. Expression of the MS2 gene is observed in tapetum of wild-type flowers at, and shortly after, the release of microspores from tetrads. The MS2 promoter controls GUS expression at a comparable stage in the tapetum of transgenic tobacco containing an MS2 promoter–GUS fusion. The occasional pollen grains produced by mutant ms2 plants have very thin pollen walls. They are also sensitive to acetolysis treatment, which is a test for the presence of an exine layer. The MS2 gene product shows sequence similarity to a jojoba protein that converts wax fatty acids to fatty alcohols. A possible function of the MS2 protein as a fatty acyl reductase in the formation of pollen wall substances is discussed.     

Glucose and phosphate toxicity in hamster preimplantation embryos involves disruption of cellular organization,including distribution of active mitochondria

While perinuclear clustering of active mitochondria, as revealed by Rhodamine 123 staining and confocal microscopy, is part of normal hamster embryo development, it is not known whether this reorganization is necessary for development. To determine if disruption of mitochondrial organization occurs in developmentally compromised embryos, the intensity of Rhodamine 123 staining was quantitated using NIH Image Software in different regions of cultured hamster 2-cell embryos exposed to either blocking (contains glucose and phosphate) or non-blocking culture conditions. Three regions within each blastomere were defined based on the organization of freshly collected embryos: cortical (ring beneath plasma membrane), perinuclear, and intermediate regions. While there was no treatment effect on the total staining intensity, glucose and phosphate treated embryos had significantly higher Rhodamine 123 staining in the intermediate region, with corresponding reduced intensity in the perinuclear region, implicating glucose and phosphate in the redistribution of mitochondria. Glucose and phosphate treatment also selectively reduced the FITC Phalloidin staining of actin microfilaments in the interior of the embryo. Neither cytochalasin D nor colchicine, at doses that blocked the second cleavage, caused redistribution of mitochondria like that seen with glucose and phosphate treatment. Additionally, cytochalasin D was unable to disrupt actin microfilaments in the perinuclear region, although it induced a “clumpy” appearance in both the mitochondria and microfilaments. This report not only offers a more mechanistic explanation of the embryo 2-cell block (translocation of mitochondria involved in glucose and phosphate inhibition) but suggests that appropriate cellular organization, including the spatial positioning of the mitochondria, may be a prerequisite for normal development and that the physical organization of the embryo is susceptible to damage by exposure to culture conditions. Mol. Reprod. Dev. 48:227–237, 1997. © 1997 Wiley-Liss, Inc.     

Mitochondrial analysis in living cells: the use of rhodamine 123 and flow cytometry

Flow cytometry combines the advantages of microscopy and biochemical analysis in a single highly sensitive technique for a rapid examination of numerous individual living cells. It has become a potent and essential tool in the studies of the physiology of the whole cell and its organelles. Rhodamine 123 is a vital fluorescent dye used in flow cytometry. As it is specifically concentrated in mitochondria because of the transmembrane potential that these organelles maintain in living cells, rhodamine 123 is thus a useful probe for monitoring the abundance and activity of mitochondria. A critical survey of the routine use of rhodamine 123 together with flow cytometry in mitochondrial research is presented.     

红菜薹雄性不育系花药败育的细胞形态学观察

采用石蜡切片技术,在光学显微镜下系统研究了红菜薹(Brassica campestris L.ssp．chinensis L.var．utilis TsenetLee．)波里马胞质雄性不育系(Polima CMS)、红菜薹萝卜胞质雄性不育系(Ogura CMS)及相应保持系花药发育过程的细胞形态学特征。观察结果表明：红菜薹Polima CMS花药发育受阻于孢原细胞阶段,不形成花粉,属无花粉型,此不育系花药不形成绒毡层和中层;而红菜薹Ogura CMS花药败育发生于小孢子母细胞期或四分体时期,表现为绒毡层细胞异常,挤压四分体,导致四分体和绒毡层同时解体而败育。     

陆地棉双隐性核雄性不育系ms5ms6花药的超微结构观察

陆地棉双隐性核雄性不育系ms₅ms₆已有较大的应用规模,但雄蕊败育的结构基础尚不明确。以核雄性不育系ms₅ms₆为材料,利用透射电镜对四分体时期和刚长出小刺突的小孢子时期的花药进行超微结构观察。发现在小孢子时期,败育花药小孢子只有外壁内层,可育小孢子此时已具有外壁外层和外壁内层了。在整个不育花药的发育过程中,不管是在小孢子还是绒毡层细胞中,内质网都异常,脂肪的积累少,这可能是导致小孢子败育最重要的原因。     

Preliminary biological evaluation of poli(amidoamine) (PAMAM) dendrimer G3.5 on selected parameters of rat liver mitochondria

Polyamidoamino (PAMAM) dendrimer of generation 3.5 (G3.5) specific interactions with rat liver mitochondria were studied. Selected parameters of mitochondria (transmembrane potential and uptake of Ca2+ ions) were investigated after the exposure to G3.5 used at the concentration of 10, 30 and 50 microM and Ca2+ ions used at 1mM. The times of preincubation of isolated liver mitochondria with dendrimer were 5, 15 and 30 min at room temperature. The mitochondrial membrane potential was monitored spectrofluorimetrically by fluorescence quenching of Rhodamine 123 (Rh 123). The changes in calcium homeostasis upon dendrimer exposure were detected using flow cytometric analysis with Fluo-3. On the one hand the obtained results revealed an impact of the tested chemical on rat liver mitochondrial function. We found that dendrimer G3.5 in a concentration above 10 microM contributes to the reduction in the transmembrane potential and hinders in the influx of Ca2+ ions to mitochondria added externally, or accelerates their efflux from mitochondria. The most effective preincubation time was observed to be 15 min for all tested concentrations. On the other hand the combined treatment of dendrimer G3.5 with Ca2+ demonstrated the protective effect of G3.5 against mitochondrial depolarization caused by calcium ions. These preliminary studies suggest that tested dendrimer can significantly affect the mitochondria and modulate their functionality.     

Postmeiotic cytokinesis and pollen aperture number determination in eudicots: effect of the cleavage wall number

Summary.  In eudicot postmeiotic tetrads, apertures are usually joined in pairs in highly conserved areas. These appear to be located at the last points of contact persisting at the end of cytokinesis between the cytoplasm of the future microspores. In order to investigate the relationship between cytokinesis and aperture formation, aperture distribution within postmeiotic tetrads and the progression of meiosis were studied in Nicotiana tabacum cv. Ambalema. This variety (inbred line) produces about 85% tricolporate pollen and 15% tetracolporate pollen grains. In addition, about 7% of tetrads are composed of four equal-sized microspores and a supernumerary pseudomicrospore of small size and an equal proportion of tetrads exhibit unpaired apertures (these apertures are not joined in pairs within tetrads). Observation of cytokinesis indicates that both unpaired apertures and pseudomicrospores could result from the persistence of late communications between microsporocytes. Observations of tetrads indicate that an increase in the number of elements that are separated during cytokinesis is correlated with an increase in microspore aperture number. All data converge to support the hypothesis that aperture site determination is partly controlled by the number of walls formed to separate the different elements of the tetrad. Received May 22, 2002; accepted October 29, 2002; published online April 2, 2003 RID="*" ID="*" Correspondence and reprints: Laboratoire de Ecologie, Systematique et Evolution, Batiment 362, Université Paris Sud, 91405 Orsay cedex, France.     

Rhodamine 123 inhibits import of rat liver mitochondrial transhydrogenase

Rhodamine 123, a laser dye, has been demonstrated to inhibit import of the precursor to pyridine dinucleotide transhydrogenase into mitochondria in rat liver cells. When rat hepatocytes were labeled with 35[S] methionine in the presence of 0.4 mM rhodamine 123, the precursor to transhydrogenase was found to have a half-life in the cytoplasm of 15 minutes as opposed to a half-life of 1-2 minutes when cells were radiolabeled in the absence of the dye. To clarify the mechanism of import inhibition, studies were initiated to assess the effect of rhodamine 123 on mitochondrial respiration. Upon addition of the dye to a mitochondrial suspension, respiration was initially enhanced, then inhibited. The inability of FCCP, a classical uncoupler, to enhance respiration during the inhibitory phase suggests that rhodamine 123 is primarily inhibiting respiration through the electron transport system rather than through the ATPase. These results suggest that rhodamine 123 may inhibit import of the transhydrogenase precursor into mitochondria by disrupting components in the mitochondrial membrane necessary for efficient import.     

Assay for apoptosis using the mitochondrial probes, Rhodamine123 and 10-N-nonyl acridine orange

Apoptosis plays a pivotal role in the regulation of cell turnover, and a defect or an excess of apoptosis has been implicated in several human diseases. Apoptosis is activated from an extracellular death signal, or from an internal pathway starting from the endoplasmatic reticulum or the mitochondria. To investigate the mitochondrial compartment during apoptosis, we have established a protocol using fluorochromes and flow cytometry to probe the structure and function of mitochondria kinetically. The protocol could be applied to whole cells or to isolated mitochondria. In the first case, cells are counterstained with ethidium bromide (EB) to evaluate plasma membrane function. The presence of the electrochemical gradient in the mitochondria is probed with Rhodamine123 (Rh123), whereas the structure and the integrity of mitochondria are assessed using 10-N-nonyl-acridine orange (NAO). Not considering the time requested for cell/mitochondria preparation and the activation of apoptosis, the protocol lasts <1 h.