Migration of DNA-Containing Organelles between Tobacco Microsporocytes during Cytomixis

Ultrastructural analysis of intercellular migration of DNA-containing organelles (nuclei, mitochondria, and plastids) in tobacco microsporogenesis during cytomixis was conducted. It was demonstrated for the first time that the migrating part of the nucleus is covered with ribosomes and can contain the accumulation of nuclear pores. The possibility of mitochondrial migration between the plant cells was proven for the first time. It was demonstrated that mitochondria extremely rarely pass into neighboring cells, and their movement occurs through one cytomictic channel. In turn, plastids can generate the accumulations around cytomictic channels and actively migrate between the cells, even through small size cytomictic channels. It was established that plastids can pass into another cell through one or several cytomictic channels, and several plastids can also simultaneously migrate through one channel. The consequences of migration of DNA-containing organelles in the cells producing the pollen are discussed.     

Cytomixis in plants: facts and doubts

The migration of nuclei between plant cells (cytomixis) is a mysterious cellular phenomenon frequently observable in the male meiosis of higher plants. Cytomixis attracts attention because of unknown cellular mechanisms underlying migration of nuclei and its potential evolutionary significance, since the genetic material is transferred between the cells that form pollen. Although cytomixis was discovered over a century ago, the advance in our understanding of this process has been rather insignificant because of methodological difficulties. The data that allowed for a new insight into this phenomenon were obtained by examining the migrating nuclei with electron and confocal laser microscopy, immunostaining, and fluorescence in situ hybridization. As has been shown, the chromatin migrating between cells is surrounded by an undamaged nuclear membrane. Such chromatin does not undergo heterochromatization and contains normal euchromatin markers. The condensation degree of the migrating chromatin corresponds to the current meiotic stage, and normal structures of synaptonemal complex are present in the migrating part of the nucleus. The cells involved in cytomixis lack any detectable morphological and molecular markers of programmed cell death. It has been shown that individual chromosomes and genomes (in the case of allopolyploids) have no predisposition to the migration between cells, i.e., parts of the nucleus are involved in cytomixis in a random manner. However, the fate of migrating chromatin after it has entered the recipient cell is still vague. A huge amount of indirect data suggests that migrating chromatin is incorporated into the nucleus of the recipient cell; nonetheless, the corresponding direct evidences are still absent. No specific markers of cytomictic chromatin have been yet discovered. Thus, the causes and consequences of cytomixis are still disputable. This review briefs the recent data on the relevant issues, describes the classical and modern methodological approaches to analysis of the intercellular migration of nuclei, and discusses the problems in cytomixis research and its prospects.     

New insights into cytomixis: specific cellular features and prevalence in higher plants

The phenomenon of intercellular migration of nuclei in plant tissues (cytomixis) was discovered over a century ago, which has been followed by numerous attempts to clarify the essence of this process as well as to determine its causes and consequences. Most attention of researchers has been paid to cytomixis in microsporogenesis, since the transfer of part of genetic material between microsporocytes may influence the ploidy level of the produced pollen and, presumably, have an evolutionary significance. This review compiles the data on cytological pattern of cytomixis and proposes a scheme as to how cytomictic channels are formed and function in angiosperms. The prevalence of cytomixis in different plant taxa is analyzed using the published data. The causes, mechanisms, and consequences of the nuclear migration between cells in plant tissues are discussed.     

An ultrastructural study of microsporogenesis in tobacco line SR1

This article provides an ultrastructural atlas of microsporogenesis in the tobacco model line SR1. The stages of cell-wall remodeling and reorganization of the intercellular channels, accompanying this process, are reported for the microspore mother cells. The meiotic changes in the cell nucleus and cytoplasm are traced. The appearance of single-, double-, or multi-membrane nuclear vacuoles in microspore mother cells and their further elimination from the nucleus are for the first time described for the genus Nicotiana as well as deviations from a normal course for this process. Intercellular chromatin migration (cytomixis) was observed in the microsporogenesis of the line SR1 and behavior of the nuclear vacuoles within the cytomictic nucleus was described for the first time. The enzymatic activity of spherosome-like vesicles in the tobacco microsporogenesis is discussed. The features of microsporogenesis in the tobacco line SR1 are compared with those of other plant species and its association with the transition from a diploid to a haploid phase of the life cycle is discussed.     

Cytomixis impairs meiosis and influences reproductive success inChlorophytum comosum (Thunb) Jacq. — An additional strategy and possible implications

Spontaneous intercellular chromatin migration/cytomixis was observed to occur in the pollen mother cells (PMCs) of theChlorophytum comosum for the first time. The migration through cytomictic channels was more pronounced in meiosis-I and very rare in meiosis-II. The process was associated with erratic meiosis, which was characterized by defects in chromosome organization and segregation. Cytomixis was more intense in the month of April than in July and consequently the frequency of meiotic irregularities was much more pronounced during the month of April. As a consequence of abnormal meiosis, fertility was drastically reduced resulting in meager seed efficiency of 17% only. Recombination system also does not guarantee the release of sufficient variability. We view the phenomenon of cytomixis as genetically controlled mechanism involving meiotic genes and operating through signal transduction pathway triggered by the environmental stimuli. The evolutionary significance and tenable hypothesis in the backdrop of existing literature is also proposed.     

Induction of cytomixis affects microsporogenesis in Sesamum indicum L. (Pedaliaceae)

Cytomixis was recorded during microsporogenesis in sesame (Sesamum indicum L.), a member of the family Pedaliaceae. The phenomenon of cytomixis was observed at various stages of meiosis in 0.5% sodium azide (SA) treated populations of Sesamum indicum L. Cytomixis was observed to occur through various methods, i.e. by forming cytoplasmic channels and direct fusion of pollen mother cells (PMCs), the former was more frequent than the latter. The migration of nuclear content involved all the chromatin/chromosomes or part of it from donor to recipient cell/cells. Some completely empty meiocytes were also observed. Stickiness, precocious movement, laggards, unorientation and micronuclei were observed in almost all the sets treated with various doses of SA. Increase in the doses of SA had a positive effect on the percentage of PMCs showing cytomixis and chromosomal abnormalities. The impact of cytomixis on meiotic behaviour, reduced pollen viability and heterogeneous sized pollen grains were observed.     

烟草花粉母细胞减数分裂前期I肌动蛋白的免疫细胞化学定位

The Pollen Mother Cells of Nicotiana Rustica L. were studied using the techniques of immunofluorescence microscopy and protein A-colloidal gold IEM. Immunofluorescence microscopy indicates the presence of actin in both nuclei and cytoplasm. IEM observation shows that gold particles are present in cytoplasm, chromatin and cytomictic channels. These results indicate that actin has some relation with chromatin condensing at synizesis. Moreover, actin may also play an important role in cytomixis.     

烟草花粉母细胞减数分裂前期Ⅰ肌动蛋白的免疫细胞化学定位(简报)

肌动蛋白是一种主要的收缩蛋白,在肌肉细胞中是广泛存在的,而在非肌肉细胞中,如在藻类细胞以及高等植物的根尖细胞、表皮细胞中,也存有肌动蛋白。在花粉萌发过程中,肌动蛋白丝集聚成束,对于花粉管的形成和受精过程有着重要作用。但已有的工作,多数限于动物细胞和植物体细胞中,关于高等植物的生殖细胞中的报道则比较少见。     

Intra- and intertissular cytomictic interactions in the microsporogenesis of mono- and dicotyledonous plants

Comparative cytological analysis of intra- and intertissular cytomictic interactions in the microsporogenesis of mono- and dicotyledonous plants has been performed for two cellular systems: the microsporocytes and the tapetum. Cytomixis was shown to be more common for intratissular interactions, and cytomixis in the tapetum exhibited taxon-specific features, both structural and temporal. Nuclear migration in the microsporocytes mostly occurred during the zygotene–pachytene and exhibited certain synchrony with cytomixis in the tapetum. Intertissular cytomictic interactions (between the tapetum and the microsporocytes) were detected only in monocotyledonous plant anthers. Intertissular interactions may reflect more intense competition for space between the tapetum and the microsporocytes during the differentiation of anther tissues. The polyploid nuclei of the tapetum and the syncytia are powerful acceptors that can compete with the microsporocytes and attract the chromatin during translocation of the latter. The absence of intertissular interactions in dicotyledonous plants may be indicative of a better balance between the processes of differentiation of somatic and generative tissues of the microsporangium as compared to monocotyledonous plants.     

EMS-induced cytomictic variability in safflower (Carthamus tinctorius L.)

Seeds of safflower (Carthamus tinctorius L.) were subjected to three treatment durations (3h, 5h and 7h) of 0.5 % Ethyl Methane Sulphonate (EMS). Microsporogenesis was carried out in the control as well as in the treated materials. EMS treated plants showed interesting feature of partial inter-meiocyte chromatin migration through channel formation, beak formation or direct cell fusion. Another interesting feature noticed during the study was the fusion among tetrads due to wall dissolution. The phenomenon of cytomixis was recorded at nearly all the stages of microsporogenesis connecting from a few to several meiocytes. Other abnormalities such as laggards, precocious movement, bridge and non-disjunction of chromosomes were also recorded but in very low frequencies. The phenomenon of cytomixis increased along with the increase in treatment duration of EMS. Cells with these types of cytomictic disturbances may probably result in uneven formation of gametes or zygote, heterogenous sized pollen grains or even loss of fertility in future.     

Role of microtubular cytoskeleton and callose walls in the manifestation of cytomixis in pollen mother cells of tobacco Nicotiana tabacum L.

The structure and dynamics of microtubular cytoskeleton and of callose walls in normal pollen mother cells (PMC) of tobacco N. tabacum L. and in cells with intercellular translocation of nuclear material (cytomictic) was studied in the course of the cell cycle. The microtubular cytoskeleton was established as playing no obvious role in the process of cytomixis. The elevated level of cytomictic seems to be due to disturbances of synthesis of callose walls as a result of their attenuation and perforation. Possible causes of cytomictic in tobacco PMC at the cellular level are discussed.     

Cytomixis in pollen mother cells of Medicago sativa L

Cytomixis (i.e., chromatin migration between meiocytes) has been detected in many plant species, but not in Medicago sativa spp. In the present study we report the identification of a few cytomictic alfalfa plants. Those plants, the "mother plants," were selfed and crossed with a normal control plant. Microsporogenesis analysis was performed on the mother plants, on the S(1) and F(1) plants, and on controls. The S(1) and F(1) plants, like the mother plants, were found to be cytomictic. Single or multiple chromatin bridges between two or more meiocytes were observed almost exclusively in prophase I. Some completely empty meiocytes were also observed. In addition to cytomixis, other meiotic abnormalities were found. Control plants showed an almost regular meiosis. The highest values of cytomixis were observed in the mother plants, and the lowest in their F(1) progenies. Variability of cytomixis in the F(1) plants is probably due to a heterozygotic condition of the parents for this trait. No significant correlation was found between cytomixis and pollen viability, even if the cytomictic plants showed low values of pollen viability.     

Efficiency of the induction of cytomixis in the microsporogenesis of dicotyledonous (<Emphasis Type="Italic">N. tabacum</Emphasis> L.) and monocotyledonous (<Emphasis Type="Italic">H. distichum</Emphasis> L.) plants by thermal stress

The efficiencies of the induction of cytomixis in microsporogenesis by thermal stress are compared in tobacco (N. tabacum L.) and barley (H. distichum L.) It has been shown that different thermal treatment schedules (budding tobacco plants at 50°C and air-dried barley grains at 48°C) produce similar results in the species: the frequency of cytomixis increases, and its maximum shifts to later stages of meiosis. However, the species show differences in response. The cytomixis frequency increase in tobacco is more pronounced, and its maximum shifts from the zygotene–pachytene stages of meiotic prophase I to prometaphase–metaphase I. Later in the meiosis, aberrations in chromosome structure and meiotic apparatus formation typical of cytomixis are noted, as well as cytomixis activation in tapetum cells. Thermal stress disturbs the integration of callose-bearing vesicles into the callose wall. Cold treatment at 7°C does not affect cytomixis frequency in tobacco microsporogenesis. Incubation of barley seeds at 48°C activates cytomixis in comparison to the control, shifts its maximum from the premeiotic interphase to zygotene, and changes the habit of cytomictic interactions from pairwise contacts to the formation of multicellular clusters. Thermal treatment induces cytomictic interactions within the tapetum and between microsporocytes and the tapetum. However, later meiotic phases show no adverse consequences of active cytomixis in barley. It is conjectured that heat stress affects callose metabolism and integration into the forming callose wall, thereby causing incomplete closure of cytomictic channels and favoring intercellular chromosome migration at advanced meiotic stages.     

EMS-induced cytomictic variability in safflower (<Emphasis Type="Italic">Carthamus tinctorius</Emphasis> L.)

Seeds of safflower (Carthamus tinctorius L.) were subjected to three treatment durations (3, 5 and 7 h) of 0.5% Ethyl Methane Sulphonate (EMS). Microsporogenesis was carried out in the control as well as in the treated materials. EMS treated plants showed interesting feature of partial inter-meiocyte chromatin migration through channel formation, beak formation or direct cell fusion. Another interesting feature noticed during the study was the fusion among tetrads due to wall dissolution. The phenomenon of cytomixis was recorded at nearly all the stages of microsporogenesis connecting from a few to several meiocytes. Other abnormalities such as laggards, precocious movement, bridge and non-disjunction of chromosomes were also recorded but in very low frequencies. The phenomenon of cytomixis increased along with the increase in treatment duration of EMS. Cells with these types of cytomictic disturbances may probably result in uneven formation of gametes or zygote, heterogenous sized pollen grains or even loss of fertility in future.     

Ultrastructure of the nuclear apparatus of the infusorian Loxodes magnus. I. The macronuclei, macronuclear anlagen and the interphasic micronuclei]

The nuclear apparatus of Loxodes magnus Stokes (Holotricha) consists of numerous macronuclei which belong to the diploid type and never divide, and of numerous micronuclei. No nuclear groups exist; individual nuclei often lie in cytoplasmic islets surrounded by large lacunae of the smooth endoplasmic reticulum. Interphasic micronuclei have two-membraned envelopes with numerous pores, usually lined at the cytoplasmic side with a layer of vacuoles, channels, or flattened vesicles of the smooth endoplasmic reticulum. The chromatin of the micronuclei consists of anastomosing threads, 0.1--0.2 mum wide, between which several nucleolus-like bodies of microfibrillar structure occur. Adult macronuclei have a similar nuclear envelope and a similar system of vacuoles, channels, and flattened agranular cisternae outside it. The macronucleus contains a single large composite nucleolus with 3 or 4 fibrillar cores inside the common granular cortex. The fibrillar cores are pierced by channels containing nucleolar organizers in the form of strands of condensed chromatin. The peripheral zone of the macronucleus is filled with decondensed chromatin fibrils and contains a number of small chromocenters and several aggregates of RNP granules. No protein inclusions (spheres) have been observed in Loxodes macronuclei. The macronuclear anlagen, developing in the cycle of every cell division, show progressive decondensation of the chromosomes and formation of several nucleoli, each with its own organizer. Later on, the nucleoli fuse into a single nucleolus. The small chromocentres are the last to form.     

Methomyl,imbraclaobrid and clethodim induced cytomixis and syncytes behaviors in PMCs of Pisum sativum L: Causes and outcomes

Cytomixis is a common phenomenon observed in meiotic cells such as anther which is influenced by various factors. Use of pesticides is a common practice in agriculture. However, it is not known whether pesticides can induce cytomixis in plant cells and induce genetic variation. To understand this, the present study was planned to assess the cytomixis and syncytes behaviors in PMCs of Pisum sativum L. Seeds of P. sativum (Family: Fabaceae) were treated with different concentrations of commonly used pesticides methomyl (ME), imbraclaobrid (IM) and clethodim (CL). Seeds were treated with various concentrations (0.1, 0.2, 0.3, 0.4 and 0.5% of ME, IM and CL prepared in water) for 1 and 3 h. Effect of pesticides on pollen fertility, frequency of cytomixis, and kind of cytomixis cells was assessed. In the cytomixis cells, the cytomictic channel (CC) and direct fusion (DF), and various stages of meiosis (PI, MI, AI and TI) with cytomixis cells were observed. In addition, frequency of syncytes cell and their various stages of meiosis I (PI, MI, AI and TI) in pollen mother cells (PMCs) was assessed. During the microsporogenesis in P. sativum, the occurrence of cytomixis and syncytes at various stages of meiosis I were seen. The formation of cytoplasmic channels and direct fusing of pollen mother cells (PMCs) were both seen to cause cytomixis, with the former being more common than the latter. The percentage of PMCs with cytomixis and syncytes cells increased with increase in the concentration of pesticides. The result of the present investigation indicates that commonly used pesticides ME, IM, and CL have a significant effect on pollen fertility, frequency of cytomixis, and kind of cytomixis cells, the cytomictic channel (CC) and direct fusion (DF), in addition, frequency of syncytes cell and their various stages of meiosis I (PI, MI, AI and TI) in pollen mother cells (PMCs) on P. sativum.     

Generation of micronuclei during interphase by coupling between cytoplasmic membrane blebbing and nuclear budding

Micronucleation, mediated by interphase nuclear budding, has been repeatedly suggested, but the process is still enigmatic. In the present study, we confirmed the previous observation that there are lamin B1-negative micronuclei in addition to the positive ones. A large cytoplasmic bleb was found to frequently entrap lamin B1-negative micronuclei, which were connected to the nucleus by a thin chromatin stalk. At the bottom of the stalk, the nuclear lamin B1 structure appeared broken. Chromatin extrusion through lamina breaks has been referred to as herniation or a blister of the nucleus, and has been observed after the expression of viral proteins. A cell line in which extrachromosomal double minutes and lamin B1 protein were simultaneously visualized in different colors in live cells was established. By using these cells, time-lapse microscopy revealed that cytoplasmic membrane blebbing occurred simultaneously with the extrusion of nuclear content, which generated lamin B1-negative micronuclei during interphase. Furthermore, activation of cytoplasmic membrane blebbing by the addition of fresh serum or camptothecin induced nuclear budding within 1 to 10 minutes, which suggested that blebbing might be the cause of the budding. After the induction of blebbing, the frequency of lamin-negative micronuclei increased. The budding was most frequent during S phase and more efficiently entrapped small extrachromosomal chromatin than the large chromosome arm. Based on these results, we suggest a novel mechanism in which cytoplasmic membrane dynamics pulls the chromatin out of the nucleus through the lamina break. Evidence for such a mechanism was obtained in certain cancer cell lines including human COLO 320 and HeLa. The mechanism could significantly perturb the genome and influence cancer cell phenotypes.     

Fine structure and cytochemistry of the nuclei of the primitive ciliateTracheloraphis crassus (Karyorelictida)

Summary The nuclei ofTracheloraphis crassus were studied using light and electron microscopy combined with Bernhard's RNP staining and pronase digestion. The nuclear apparatus of this species consists of a longitudinal row of 11–43 macronuclei and 4–16 micronuclei. Like in all karyorelictids, the macronuclei are unable to divide and become segregated during cytokinesis; their number is supplemented in every cell cycle by differentiation of several new macronuclei from micronuclei.Each adult macronucleus contains a single compact endonuclear aggregate of several large chromocenters, readily destained with EDTA, and several RNP containing nucleoli. There is continuity between the material of the chromocenters and the decondensed DNP fibrils in the nuclear matrix. The nucleoli contain NORs in the form of fibrillar centers. The endonuclear aggregate includes also groups of RNP granules which are especially resistant to EDTA destaining. A microfibrillar sphere, usually localized at the periphery of the aggregate, contacts one or several nucleoli. The sphere is not bleached with EDTA, and only its periphery becomes digested with pronase. The macronuclear matrix consists of both protein fibrils and pronase-resistant fibrils, the latter being localized at the nuclear periphery.Developing macronuclear primordia contain loose strands of decondensed chromatin; only later they form chromocenters and nucleoli.The micronuclei reproduce by mitosis with typical chromosomes (2n=66). During interphase, they are filled with condensed chromatin which can be bleached with EDTA; they form no nucleoli. Ring-like lamellae, existing in the cavities of the chromatin mass, stain for RNA (after Bernhard) and are pronase-sensitive. These lamellae resemble the kinetochore material conserved during interphase in another karyorelictid ciliate,Trachelocerca geopetiti.     

Maternal effect mutations affecting macronuclear determination and development in Paramecium tetraurelia

Gene mutations that interfere with macronuclear development in Paramecium were obtained by selecting lines that failed to produce normal macronuclear anlagen following the second autogamy after mutagenesis. The mutants fell into several complementation groups. There was one case of apparent intragenic noncomplementation among the eight mutants examined. In the stronger mutants, macronuclear anlagen were not formed, and all four mitotic products of the posfzygotic divisions of the synkaryon remained as micronuclei. Under semirestrictive conditions, cells often contained a single anlage, suggesting that determination of anlagen was a discrete event for each nucleus. The missing anlagen trait was recessive and associated with a strong maternal effect. The phenocritical period of one of the stronger alleles, aal^a, began at the second postzygotic division and ended with the first morphological differentiation of macronuclear anlagen. Nuclear migration in this mutant was abnormal. Under restrictive conditions, the posterior products of the second postzygotic division reached a posterior-most position, which was 8% of cell length more anterior than that of the most posterior nuclei in wild-type cells. Under permissive conditions, the pattern of migration was intermediate between that of wild-type cells and mutants under fully restrictive conditions. The patterns of nuclear migration were consistent with the nuclear growth kinetics.     

Ultrastructural Observation on the Intra- and Intercellular Microtrabecular Network of the Pollen Mother Cells in Onion (Allium cepa)

Using DGD embedment-free electron microscopy, ultrastructural observation on the intra- and intercellular microtrabecular network (MN) of the pollen mother cells (PMC) of the whole meiotic prophase Ⅰ in onion ( Allium cepa L.) was performed. Complex nuclear MN was observed in the nucleus of PMCs, spreading throughout the nuclear region. The nucleolus and chromosomes were connected with the MN filament network. The uniformity of nuclear MN changed with the development of the PMCs. A lamina-like structure surrounded the nucleus and joined the MN in nucleus and in cytoplasm, but it disassembled at the end of prophase Ⅰ. There was also a complex cytoplasmic MN in PMCs, without obvious variation during the prophase Ⅰ. Furthermore, MN in cytoplasmic connections (plasmodesmata and cytoplasmic channels) was noticed to link the frameworks in two neighboring PMCs into one entity. Cytomixis was observed at synizesis of prophase Ⅰ in this experiment, and MN in cytoplasm and in nucleus was noticed to distribute in these granules which migrated from one PMC into its neighboring cell. At this time the nucleus moved aside from center of the PMC, but the rest of the cell was still fulfilled with MN filaments. The relationships of nuclear MN with nucleolus and chromosomes, lamina with nucleus, as well as intra- and intercellular MN with cytomixis are discussed in this paper.