Vegetative Nucleus Research Articles

Ultrastructure of fertilization in Plumbago zeylanica

The synergidless female gametophyte of <em>Plumbago zeylanica</em> receives the pollen tube through specialized cell wall ingrowths at the base of the egg; tube growth continues between egg and central cells. Pollen tube discharge occurs between egg and central cell and results in release of two male gametes, vegetative nucleus, and some pollen cytoplasm. Except for the location of gamete discharge, details of transmission and fusion of gametic nuclei appear to conform to reports of these processes in taxa possessing conventional embryo sacs.

Autoradiographic studies of DNA and histone synthesis in successive differentiation stages of pollen grain in Hyacinthus orientalis L.

DNA and histone synthesis in five consecutive morphological stages of <em>Hyacinthus orientalis</em> L. pollen grain differentiation were studied autoradiographically. DNA synthesis was found to occur in both the generative and the vegetative cell. DNA replication in the generative cell took place when the generative cell was still adhered to the pollen grain wall but already devoid of callose wall. DNA synthesis in the generative cell slightly preceded that in the vegetative cell. Histones were synthesized in phase S of the generative and vegetative cell. In the generative cell histone synthesis also continued at a lower level after completion of DNA replication. In the developmental stages under study the nuclei of the generative cells were decidedly richer in lysine histones than vegetative cell nuclei.

Ultrastructural transformations of nuclei in differentiating Hyacirlthus orientalis L. pollen grain cells

The ultrastructure of the pollen nuclei was studied during pollen maturation in <em>Hyacinthus orientalis</em> L., in which both pollen cells go through full interphase (G<sub>1</sub> S, G<sub>2</sub>). The particular stages of interphase, however, do not proceed simultaneously in the vegetative and generative nuclei. The ultrastructural transformations of both pollen nuclei were analysed with reference to the dynamics of variations in the level of RNA and protein synthesis, investigated earlier. A distinction is drawn between the structural changes common for both nuclei, linked immanently with the interphase and the transformations connected with the differentiation of pollen cells.

Pollen grain of barley (Hordeum vulgare L.) - pattern of development

Pollen development in barley follows the general pattern established for other species of <em>Poaceae</em>: 1) microspore division occurs at the vacuolate microspore stage with polarly located nucleus; 2) microspore mitosis is immediately followed by phragmoplast and cell plate formation; 3) in consequence or unequal microspore division, the generative cell, at first attached to the pollen wall, is separated from the vegetative cell by a callosic wall; 4) during the postmitotic two-cell stage of development, the vegetative nucleus migrates to the aperture pole and is followed by the generative cell that is detached and free of callose wall. In this position the generative cell divides into two sperm cells. These data do not confirm the interpretation of pollen grain development in barley given by Cass and Karas in Can. J. Bot. 53: 1051-1062, 1975.

Localization of actin in pollen tubes of Ornithogalum virens L.

The germinating pollen grain (in vivo on the stigma or in vitro in germination medium) forms a pollen tube which transports the vegetative nucleus and generative cell/two sperm cells participating in the process of double fertilization. The growth of the tube and the transport of organelles and the cells occur due to two major motor systems existing in the pollen tubes of higher plants: the tubuline-dynein/kinesin and the actin-myosin system. In pollen tubes of <em>Ornithogalum virens</em> the actin filaments were labelled with TRITC-phalloidin (2 µg/ml) in the PIPES buffer and the 10% sucrose, without the fixative and DMSO. Omission of the fixative and permeabilizing agent (DMSO) allowed better preservation of the structure, and the "fluorescence" of actin was observed in living pollen tubes. Observations in CLSM (confocal laser scanning microscope) showed that actin is distributed in the vicinity of the cell membrane. This could support the view that actin filaments and the plasmalemma form the pollen tube cortex along which the cytoplasmic movement of organelles, and cell transport occurs.

Chromatin Alterations during Pollen Development in Hordeum vulgare

The dynamics of posttranslational histone modifications in relation to nuclear architecture has been analyzed during pollen development in Hordeum vulgare L. cv. Igri. Notwithstanding the asymmetry of cytokinesis associated with pollen mitosis I, immunolabeling revealed that the vegetative and generative nuclei initially display identical chromatin modification patterns. Yet, differential chromatin modification patterns between vegetative and generative nuclei emerge with the development of conspicuous differences in nuclear morphology as visualized by 4′,6-diamidino-2-phenylindole staining. The temporal and spatial distribution of most histone modifications observed is in agreement with reduced gene activity in the generative nucleus and increased expression in the vegetative nucleus as indicated by immunolabeling of active RNA polymerase II. Signals of trimethylation of histone H3 lysine 27 proved to be particularly enriched in euchromatic domains of subtelomeric regions. In the context of nuclear differentiation in bicellular pollen, this modification became restricted to the vegetative nucleus, indicating a role in activating rather than suppressing gene expression. The presence of acetylated histone H3 at lysine 9 in the cytoplasm of the generative cell is indicative of a more complex, still unknown function of this particular modification.

The Arabidopsis general transcription factor TFIIB1 (AtTFIIB1) is required for pollen tube growth and endosperm development

Pollen tube growth and endosperm development are important for fertilization and seed formation. The genetic mechanism of the processes remains poorly understood. This study reports the functional characterization of AtTFIIB1 in pollen tube growth and endosperm development. AtTFIIB1 shares 86% and 44% similarity with AtTFIIB2 and AtTFIIB3/AtpBRP2, respectively. It is expressed in many tissues including vegetative nuclei and generative cells of pollen grains and pollen tubes, endosperm, and embryos. It is thus different from AtTFIIB2, whose expression is not found in the endosperm and vegetative nucleus of mature pollen, and AtTFIIB3/AtpBRP2, which is expressed mostly in male gametophytes and weakly in seeds. Mutations in AtTFIIB1 caused a drastic retardation of pollen tube growth and endosperm development, as well as impaired pollen tube guidance and reception, leading to disruption of fertilization and seed development. Expression of AtTFIIB2 driven by the AtTFIIB1 promoter could restore the defective pollen tube growth, guidance, and reception completely, but only partially recovered the seed development in attfiib1, whilst expression of AtTFIIB3/AtpBRP2 driven by the AtTFIIB1 promoter could rescue only the defective attfiib1 seeds. All these results suggest that AtTFIIB1 plays important roles in pollen tube growth, guidance, and reception as well as endosperm development and is partially functionally different from AtTFIIB2 and AtTFIIB3/AtpBRP2.

New insights into Fe localization in plant tissues

Deciphering cellular iron (Fe) homeostasis requires having access to both quantitative and qualitative information on the subcellular pools of Fe in tissues and their dynamics within the cells. We have taken advantage of the Perls/DAB Fe staining procedure to perform a systematic analysis of Fe distribution in roots, leaves and reproductive organs of the model plant Arabidopsis thaliana, using wild-type and mutant genotypes affected in iron transport and storage. Roots of soil-grown plants accumulate iron in the apoplast of the central cylinder, a pattern that is strongly intensified when the citrate effluxer FRD3 is not functional, thus stressing the importance of citrate in the apoplastic movement of Fe. In leaves, Fe level is low and only detected in and around vascular tissues. In contrast, Fe staining in leaves of iron-treated plants extends in the surrounding mesophyll cells where Fe deposits, likely corresponding to Fe-ferritin complexes, accumulate in the chloroplasts. The loss of ferritins in the fer1,3,4 triple mutant provoked a massive accumulation of Fe in the apoplastic space, suggesting that in the absence of iron buffering in the chloroplast, cells activate iron efflux and/or repress iron influx to limit the amount of iron in the cell. In flowers, Perls/DAB staining has revealed a major sink for Fe in the anthers. In particular, developing pollen grains accumulate detectable amounts of Fe in small-size intracellular bodies that aggregate around the vegetative nucleus at the binuclear stage and that were identified as amyloplasts. In conclusion, using the Perls/DAB procedure combined to selected mutant genotypes, this study has established a reliable atlas of Fe distribution in the main Arabidopsis organs, proving and refining long-assumed intracellular locations and uncovering new ones. This “iron map” of Arabidopsis will serve as a basis for future studies of possible actors of iron movement in plant tissues and cell compartments.

A method to isolate male gametic nuclei from pear pollen tubes

SummaryThe nucleus plays an essential role in regulating most biological activities in most living organisms. Pear (Pyrus bretshneideri) pollen is binucleate, possessing a vegetative nucleus and a generative nucleus. To reveal the nuclear proteome and to produce accurate physical maps of DNA sequences using fluorescence in situ hybridisation (FISH) analysis, it is essential to obtain high quality nuclei. Protocols to isolate intact nuclei usually involve liberating them from centrifuged cells, then filtering them using a nylon mesh. Releasing the nuclei is the most critical step to obtain high quality material from small amounts of tissue. Here, we describe an easy and efficient method to isolate and purify both the generative and vegetative nuclei from pear pollen tubes. The nuclei were liberated by grinding pollen tubes, filtered on a cell strainer with a pore size of 40 µm, then passing them through a second cell strainer with a mesh size of 25 µm. After these procedures, and purification through a 28% (v/v) PercollTM gradient by centrifugation, the isolated nuclei exhibited a high level of purity and integrity, with little debris, as confirmed using enzyme markers and 4’,6-diamidino-2-phenylindole (DAPI) staining. The purified nuclei were used to prepare nuclear proteins and DNA fibres. The present protocol proved suitable for the isolation of male gametic nuclei from in vitro-cultivated pear pollen tubes, for nuclear proteome analysis, and for the preparation of DNA fibres for FISH analysis.

FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei

BackgroundThe male germline in flowering plants differentiates by asymmetric division of haploid uninucleated microspores, giving rise to a vegetative cell enclosing a smaller generative cell, which eventually undergoes a second mitosis to originate two sperm cells. The vegetative cell and the sperm cells activate distinct genetic and epigenetic mechanisms to control pollen tube growth and germ cell specification, respectively. Therefore, a comprehensive characterization of these processes relies on efficient methods to isolate each of the different cell types throughout male gametogenesis.ResultsWe developed stable transgenic Arabidopsis lines and reliable purification tools based on Fluorescence-Activated Cell Sorting (FACS) in order to isolate highly pure and viable fractions of each cell/nuclei type before and after pollen mitosis. In the case of mature pollen, this was accomplished by expressing GFP and RFP in the sperm and vegetative nuclei, respectively, resulting in 99% pure sorted populations. Microspores were also purified by FACS taking advantage of their characteristic small size and autofluorescent properties, and were confirmed to be 98% pure.ConclusionsWe provide simple and efficient FACS-based purification protocols for Arabidopsis microspores, vegetative nuclei and sperm cells. This paves the way for subsequent molecular analysis such as transcriptomics, DNA methylation analysis and chromatin immunoprecipitation, in the developmental context of microgametogenesis in Arabidopsis.

Anther structure and pollen development in Melicoccus lepidopetalus (Sapindaceae): An evolutionary approach to dioecy in the family

Anther and pollen development in staminate and pistillate flowers of dioecious Melicoccus lepidopetalus (Sapindaceae) were examined by light and electron microscopy. Young anthers are similar in both types of flowers; they consist of epidermis, endothecium, two to four middle layers and a secretory tapetum. The microspore tetrads are tetrahedral. The mature anther in staminate flowers presents compressed epidermal cells and endothecium cells with fibrillar thickenings. A single locule is formed in the theca by dissolution of the septum and pollen grains are shed at two-celled stage. The mature anthers of pistillate flowers differ anatomically from those of staminate flowers. The epidermis is not compressed, the endothecium does not develop fibrillar thickenings, middle layers and tapetum are generally persisting, and the stomium is nonfunctional. Microspore degeneration begins after meiosis of microspore mother cells. At anthesis, uninucleate microspores and pollen grains with vegetative and generative nuclei with no cytokinesis are observed. Some pollen walls display an abnormal exine deposition, whereas others show a well formed exine, although both are devoid of intine. These results suggest that in the evolution towards unisexuality, the developmental differences of anther wall tissues and pollen grains between pistillate and staminate flowers might become more pronounced in a derived condition, such as dioecy.

Reprogramming of DNA Methylation in Pollen Guides Epigenetic Inheritance via Small RNA

Epigenetic inheritance is more widespread in plants than in mammals, in part because mammals erase epigenetic information by germline reprogramming. We sequenced the methylome of three haploid cell types from developing pollen: the sperm cell, the vegetative cell, and their precursor, the postmeiotic microspore, and found that unlike in mammals the plant germline retains CG and CHG DNA methylation. However, CHH methylation is lost from retrotransposons in microspores and sperm cells and restored by de novo DNA methyltransferase guided by 24 nt small interfering RNA, both in the vegetative nucleus and in the embryo after fertilization. In the vegetative nucleus, CG methylation is lost from targets of DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), and their homologs, which include imprinted loci and recurrent epialleles that accumulate corresponding small RNA and are premethylated in sperm. Thus genome reprogramming in pollen contributes to epigenetic inheritance, transposon silencing, and imprinting, guided by small RNA.

Distribution of nuclei and microfilaments during pollen germination in Populus tomentosa Carr.

Pollen tubes transport nuclei to the ovules for fertilization. The distribution of microfilaments and the nuclei were investigated by fluorescent phalloidin labeling and DAPI (4', 6-diamidino-2- phenylindole) during pollen germination and pollen tube growth of Populus tomentosa Carr., a Chinese native tree species. The coexistence of trinucleate and binucleate pollen was confirmed, which had been previously considered binucleate. Three distinct typical microfilament structures were found, and F-actin was present at the periphery of both the vegetative and the sperm nuclei. The investigation indicated that movement of sperm nuclei and vegetative nucleus is related to the microfilament system. Key words: Generative nucleus, vegetative nucleus, actin cytoskeleton, pollen tube growth.

Reprogramming the Epigenome in Arabidopsis Pollen

Epigenetic reprogramming in Arabidopsis thaliana occurs in developing pollen. The male gametophyte is derived from haploid microspores via two postmeiotic cell divisions to give rise to the gametes (sperm cells, SC) and the vegetative cell (VC). The purification of individual cell types during pollen development coupled with genome-wide DNA methylation analysis and small RNA sequencing has revealed a dynamic regulation of the epigenome during gametogenesis. Interestingly, imprinted loci and previously identified variable epialleles are hypermethylated in the germline; however, their stability after fertilization appears to require targeted demethylation in the neighboring vegetative cell nucleus, possibly by releasing mobile small RNAs that reinforce transcriptional gene silencing and DNA methylation in the gametes. These results have led to a new model for the establishment and transgenerational maintenance of epigenetic marks in flowering plants.

Male gametogenesis ultrastructure of Codium fragile subsp. novae-zelandiae (Bryopsidophyceae, Chlorophyta)

Miravalles A.B., Leonardi P.I. and Cáceres E.J. 2011. Male gametogenesis ultrastructure of Codium fragile subsp. novae-zelandiae (Bryopsidophyceae, Chlorophyta). Phycologia 50: 370–378. DOI: 10.2216/10-25.1Male gametogenesis was studied by light and electron microscopy in the dioecious siphonous green alga Codium fragile subsp. novae-zelandiae. Gametangia were formed on the sides of utricules. Initially, gametangia showed vegetative interphase nuclei and chloroplasts already with a mature structure with few thylakoid lamellae and a single starch granule. Then, mitotic divisions occurred, originating a proliferation of gametic nuclei. Features of the gametangial mitosis were similar to vegetative mitoses of other Bryopsidophyceae. The fusion of minute vesicles clearly delimited uninucleate portions of protoplasm with a large mitochondrion and chloroplasts. Ultimately, the gamete final shape was delimited, and the basal-flagellar apparatus developed. Mature gametes were liberated immersed in a mucilaginous material throughout an apical opening. They were elongated-ovoid in shape and showed two flagella, a hyaline anterior portion and a rear portion with two or three yellow-green chloroplasts. The nucleus was anterior, with abundant condensed chromatin and without nucleolus. A large ‘inverted-V-shaped’ mitochondrion was located in the anterior region of the gamete. Chloroplasts had poorly developed lamellae and a large starch granule. The basal-flagellar apparatus features were the typical for the class.

Spatial and Temporal Expression of Actin Depolymerizing Factors ADF7 and ADF10 during Male Gametophyte Development in Arabidopsis thaliana

The actin cytoskeleton plays a crucial role in many aspects of plant cell development. During male gametophyte development, the actin arrays are conspicuously remodeled both during pollen maturation in the anther and after pollen hydration on the receptive stigma and pollen tube elongation. Remodeling of actin arrays results from the highly orchestrated activities of numerous actin binding proteins (ABPs). A key player in actin remodeling is the actin depolymerizing factor (ADF), which increases actin filament treadmilling rates. We prepared fluorescent protein fusions of two Arabidopsis pollen-specific ADFs, ADF7 and ADF10. We monitored the expression and subcellular localization of these proteins during male gametophyte development, pollen germination and pollen tube growth. ADF7 and ADF10 were differentially expressed with the ADF7 signal appearing in the microspore stage and that of ADF10 only during the polarized microspore stage. ADF7 was associated with the microspore nucleus and the vegetative nucleus of the mature grain during less metabolically active stages, but in germinating pollen grains and elongating pollen tubes, it was associated with the subapical actin fringe. On the other hand, ADF10 was associated with filamentous actin in the developing gametophyte, in particular with the arrays surrounding the apertures of the mature pollen grain. In the shank of elongating pollen tubes, ADF10 was associated with thick actin cables. We propose possible specific functions of these two ADFs based on their differences in expression and localization.

Gametophytic development of Brassica napus pollen in vitroenables examination of cytoskeleton and nuclear movements

Isolated microspores and pollen suspension of Brassica napus “Topas” cultured in NLN-13 medium at 18°C follow gametophytic pathway and develop into pollen grains closely resembling pollen formed in planta. This culture system complemented with whole-mount immunocytochemical technology and novel confocal laser scanning optical technique enables detailed studies of male gametophyte including asymmetric division, cytoskeleton, and nuclear movements. Microtubular cytoskeleton configurationally changed in successive stages of pollen development. The most prominent role of microtubules (MTs) was observed just before and during nuclear migration at the early and mid-bi-cellular stage. At the early bi-cellular stage, parallel arrangement of cortical and endoplasmic MTs to the long axis of the generative cell (GC) as well as MTs within GC under the plasmalemma bordering vegetative cell (VC) were responsible for GC lens shape. At the beginning of the GC migration, endoplasmic microtubules (EMTs) of the VC radiated from the nuclear envelope. Most cortical and EMTs of the VC were found near the sporoderm. At the same time, pattern of MTs observed in GC was considerably different. Multiple EMTs of the GC, previously parallel aligned, reorganized, and start to surround GC, forming a basket-like structure. These results suggest that EMTs of GC provoke changes in GC shape, its detachment from the sporoderm, and play an important role in GC migration to the vegetative nucleus (VN). During the process of migration of the GC to the VC, multiple and thick bundles of MTs, radiating from the cytoplasm near GC plasma membrane, arranged perpendicular to the narrow end of the GC and organized into a “comet-tail” form. These GC “tail” MTs became shortened and the generative nucleus (GN) took a ball shape. The dynamic changes of MTs accompanied polarized distribution pattern of mitochondria and endoplasmic reticulum. In order to confirm the role of MTs in pollen development, a “whole-mount” immunodetection technique and confocal laser-scanning microscopy was essential.Electronic supplementary materialThe online version of this article (doi:10.1007/s00709-011-0287-0) contains supplementary material, which is available to authorized users.

CENH3 distribution and differential chromatin modifications during pollen development in rye (Secale cereale L.)

Microgametogenesis in angiosperms results in two structurally and functionally different cells, one generative cell, which subsequently forms the sperm cells, and the vegetative cell. We analysed the chromatin properties of both types of nuclei after first and second pollen mitosis in rye (Secale cereale). The condensed chromatin of generative nuclei is earmarked by an enhanced level of histone H3K4/K9 dimethylation and H3K9 acetylation. The less condensed vegetative nuclei are RNA polymerase II positive. Trimethylation of H3K27 is not involved in transcriptional downregulation of genes located in generative nuclei as H3K27me3 was exclusively detected in the vegetative nuclei. The global level of DNA methylation does not differ between both types of pollen nuclei. In rye, unlike in Arabidopsis thaliana (Ingouff et al. Curr Biol 17:1032-1037 2007; Schoft et al. EMBO Rep 10:1015-1021 2009), centromeric histone H3 is not excluded from the chromatin of the vegetative nucleus and the condensation degree of centromeric and subtelomeric regions did not differ between the generative and vegetative nuclei. Differences between rye and A. thaliana data suggest that the chromatin organization in mature nuclei of pollen grains is not universal across angiosperms.

Cytoplasmic connection of sperm cells to the pollen vegetative cell nucleus: potential roles of the male germ unit revisited

The male germ cells of angiosperm plants are neither free-living nor flagellated and therefore are dependent on the unique structure of the pollen grain for fertilization. During angiosperm male gametogenesis, an asymmetric mitotic division produces the generative cell, which is completely enclosed within the cytoplasm of the larger pollen grain vegetative cell. Mitotic division of the generative cell generates two sperm cells that remain connected by a common extracellular matrix with potential intercellular connections. In addition, one sperm cell has a cytoplasmic projection in contact with the vegetative cell nucleus. The shared extracellular matrix of the two sperm cells and the physical association of one sperm cell to the vegetative cell nucleus forms a linkage of all the genetic material in the pollen grain, termed the male germ unit. Found in species representing both the monocot and eudicot lineages, the cytoplasmic projection is formed by vesicle formation and microtubule elongation shortly after the formation of the generative cell and tethers the male germ unit until just prior to fertilization. The cytoplasmic projection plays a structural role in linking the male germ unit, but potentially plays other important roles. Recently, it has been speculated that the cytoplasmic projection and the male germ unit may facilitate communication between the somatic vegetative cell nucleus and the germinal sperm cells, via RNA and/or protein transport. This review focuses on the nature of the sperm cell cytoplasmic projection and the potential communicative function of the male germ unit.

Isolated microspore culture of chickpea (Cicer arietinum L.): induction of androgenesis and cytological analysis of early haploid divisions

Routine production of haploid plants has not been reported for any legume, despite the major role these species play in sustainable farming systems and human nutrition. It is within this context that we report a protocol for the induction of haploid development in chickpea (Cicer arietinum L.) using isolated microspore culture. The cultivars “Rupali”, “Narayen”, and “Kimberley Large” were identified as responsive to isolated microspore culture. Flower bud length and microspore developmental stage were correlated for these cultivars. Depending on the cultivar, buds 2.85–3.5 mm in length contained uninucleate microspores. Microspores from donor plants grown in winter and spring were more responsive than those grown in summer. A cold treatment (4°C) of between 24 and 48 h enhanced microspore response in winter- and spring-grown material but was not effective in summer-grown material. A medium developed by the authors was effective for microspore induction and early-stage embryo development. The addition of hormones to this medium was promotive of microspore induction in winter- and spring-grown material, but not in summer-grown material. The initial haploid division predominantly occurred via symmetrical division of the vegetative nucleus. Further research is under way to convert pro-embryos into plants.