Cytophotometric study of nuclear differentiation during pollen development in Tradescantia Paludosa

Summary During pollen development the dry weight, total protein, histone, DNA, arginine, and lysine content were analysed by cytophotometric methods in partially isolated nuclei. The amount of analysed substances increased from the end of the meiosis to the mitosis of the microspores to the double of the initial values. After mitosis the ratio histone/DNA remained almost unchanged in both vegetative and generative nuclei. On the other hand a large difference in the ratio non-histone protein/DNA could be observed, the vegetative nucleus containing more non-histone protein than the generative nucleus. The rate of RNA synthesis being higher in the vegetative nuclei, these non-histone proteins may have some function in nuclear activation. The DNA of the generative nucleus is duplicated before anthesis, whilst in the vegetative nucleus the DNA content remains constant.     

Assorbimento dopo colorazione al Fast-green di cellule della caliptra di Allium cepa localizzate in differenti regioni istologiche

Abstract

Fast-green absorption of cells localized in different regions of Allium cepa root cap.—The nuclear and cytoplasmic protein content of two cell population (C2 = columella cells; P2 = peripheral cells) has been determined by Barr and Stroud cytophotometer after Fast-green staining (after DNA extraction and without DNA extraction). The collected data seem indicate that: 1) histone content is higher in nuclei of C2 cells than in nuclei of P2 ones; 2) acid proteins content is higher in nuclei of P2 cells than in those of C2 ones; 3) the cytoplasmic proteins of the two cell populations are quite different in their Fast-green reactivity.     

Uno studio citofotometrico delle proteine durante la mitosi nel meristema radicale di Allium cepa L.

Abstract

A cytophotometric study of proteins during mitosis in root tip meristematic cells of «Allium cepa L.». — Spectrophotometric analyses of the amount of Fast-green stainable proteins at different pH values (8,1; 6; 4; 2) have been accomplished in Allium cepa root tip meristematic cells during the four phases of mitosis. The results seem to indicate that: a) the highest absorption is detectable in correspondence of metaphase at each of the four pH values; b) the transition from prophase to metaphase is characterized by an increase of both proteins reacting at pH 8,1 (histones) and between pH 6 and pH 8,1 (neutral proteins); c) the transition from metaphase to anaphase is characterized by a loss of histones and of proteins reacting between pH 4 and pH 6 (acid proteins); d) the transition from anaphase to telophase is characterized by a loss of neutral proteins. The data are discussed in relation to the problem of chromosome coiling.     

The developmental fate of angiosperm pollen is associated with a preferential decrease in the level of histone H1 in the vegetative nucleus

In angiosperm pollen, the vegetative cell is assumed to function as a gametophytic cell in pollen germination and growth of the pollen tube. The chromatin in the nucleus of the vegetative cell gradually disperses after microspore mitosis, whereas the chromatin in the nucleus of the other generative cell remains highly condensed during the formation of two sperm nuclei. In order to explain the difference in chromatin condensation between the vegetative and generative nuclei, we analyzed the histone composition of each nucleus in Lilium longiflorum Thunb. and Tulipa gesneriana immunocytochemically, using specific antisera raised against histones H1 and H2B of Lilium. We found that the level of histone H1 decreased gradually only in the vegetative nucleus during the development of pollen within anthers and that the vegetative nucleus in mature pollen after anther dehiscence contained little histone H1. By contrast, the vegetative nucleus contained the same amount or more of histone H2B than the generative nucleus. The preferential decrease in the level of histone H1 occurred in anomalous pollen with one nucleus (uninucleate pollen) or with two similar nuclei (equally divided pollen), which had been induced by treatment with colchicine. The nuclei in the anomalous pollen resembled vegetative nuclei in terms of structure and staining properties. The anomalous pollen was able to germinate and extend a pollen tube. From these results, it is suggested that the preferential decrease in level of histone H1 in pollen nuclei is essential for development of the male gametophytic cell through large-scale expression of genes that include pollen-specific genes, which results in pollen germination and growth of the pollen tube. Received: 9 May 1998 / Accepted: 4 June 1998     

AN AUTORADIOGRAPHIC STUDY OF RNA SYNTHESIS DURING POLLEN EMBRYOGENESIS IN HYOSCYAMUS NIGER (HENBANE)

RNA synthesis during pollen embryogenesis in cultured anther segments of Hyoscyamus niger (henbane) has been followed by autoradiography of ³H-uridine incorporation. Embryogenic divisions were initiated in binucleate pollen grains in which the generative nucleus or both generative and vegetative nuclei synthesized RNA. When the first haploid mitosis in culture resulted in pollen grains with two nearly identical nuclei, those in which both nuclei synthesized RNA became embryogenic. Binucleate pollen grains in which ³H-uridine incorporation was confined exclusively to the vegetative nucleus gradually became starch-filled and nonembryogenic. Based on the degree of involvement of the vegetative nucleus in embryoid formation, some differences were noted between the counts of autoradiographic silver grains over cells cut off by the generative and vegetative nuclei during progressive embryogenesis. The possible significance of RNA synthesis in the nuclei of binucleate pollen grains in determining the pathway of embryogenic divisions is discussed.     

Nuclear DNA synthesis during the induction of embryogenesis in cultured microspores and pollen of Brassica napus L.

The dynamics of nuclear DNA synthesis were analysed in isolated microspores and pollen of Brassica napus that were induced to form embryos. DNA synthesis was visualized by the immunocytochemical labelling of incorporated Bromodeoxyuridine (BrdU), applied continuously or as a pulse during the first 24 h of culture under embryogenic (32 °C) and non-embryogenic (18 °C) conditions. Total DNA content of the nuclei was determined by microspectrophotometry. At the moment of isolation, microspore nuclei and nuclei of generative cells were at the G1, S or G2 phase. Vegetative nuclei of pollen were always in G1 at the onset of culture. When microspores were cultured at 18 °C, they followed the normal gametophytic development; when cultured at 32 °C, they divided symmetrically and became embryogenic or continued gametophytic development. Because the two nuclei of the symmetrically divided microspores were either both labelled with BrdU or not labelled at all, we concluded that microspores are inducible to form embryos from the G1 until the G2 phase. When bicellular pollen were cultured at 18 °C, they exhibited labelling exclusively in generative nuclei. This is comparable to the gametophytic development that occurs in vivo. Early bicellular pollen cultured at 32 °C, however, also exhibited replication in vegetative nuclei. The majority of vegetative nuclei re-entered the cell cycle after 12 h of culture. Replication in the vegetative cells preceded division of the vegetative cell, a prerequisite for pollen-derived embryogenesis.     

Nuclear and cell migration during pollen development in rice (Oryza sativa L.)

Nuclear and cell migration during pollen development in rice were studied using semi-thin section light microscopy, differential interference contrast microscopy and epifluorescence microscopy. Four migrations of nuclei and cells were observed and described in detail here. The first nuclear migration occurs at the uninucleate microspore stage, when the nucleus of the microspore migrates from the center to the periphery of the cell, and then to the wall opposite the pollen aperture where pollen mitosis I takes place. The second migration occurs at the early bicellular pollen stage, with the vegetative nucleus migrating three-quarters of the circumference of the pollen wall, finally locating at the periphery of the wall where the microspore cell nucleus is positioned. The third migration occurs at the late bicellular pollen stage, with the vegetative nucleus migrating from the periphery of the cell to the central part of the pollen and the generative cell migrating from the opposite side of the aperture to a position between the aperture and the vegetative nucleus where pollen mitosis II takes place. The fourth migration appears at the mature pollen stage when the two sperm cells and the vegetative nucleus migrate to the opposite side of the aperture, finally becoming positioned in the cytoplasm of the vegetative cell distal to the aperture where the male germ unit forms. Cytological observations of pollen abortion resulting from allelic interaction at the S-a, S-b and S-c loci show that abnormalities in the first or second nuclear migration result in the formation of empty abortive pollen, whereas abnormalities in the third or fourth migrations cause production of stainable abortive pollen.     

Epigenetic marks in the mature pollen of Quercus suber L. (Fagaceae)

We have analysed the distribution of epigenetic marks for histone modifications at lysine residues H3 and H4, and DNA methylation, in the nuclei of mature pollen cells of the Angiosperm tree Quercus suber; a monoecious wind pollinated species with a protandrous system, and a long post-pollination period. The ultrasonic treatment developed for the isolation of pollen nuclei proved to be a fast and reliable method, preventing the interference of cell wall autofluorescence in the in situ immunolabelling assays. In contrast with previous studies on herbaceous species with short progamic phases, our results are consistent with a high level of silent (5-mC and H3K9me2) epigenetic marks on chromatin of the generative nucleus, and the prevalence of active marks (H3K9me3 and H4Kac) in the vegetative nucleus. The findings are discussed in terms of the pollination/fertilization timing strategy adopted by this plant species.     

Behavior of organelle nuclei (nucleoids) in generative and vegetative cells during maturation of pollen inLilium longiflorum andPelargonium zonale

Summary The behavior of organelle nuclei during maturation of the male gametes ofLilium longiflorum andPelargonium zonale was examined by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and Southern hybridization. The organelle nuclei in both generative and vegetative cells inL. longiflorum were preferentially degraded during the maturation of the male gametes. In the mature pollen grains ofL. longiflorum, there were absolutely no organelle nuclei visible in the cytoplasm of the generative cells. In the vegetative cells, almost all the organelle nuclei were degraded. However, in contrast to the situation in generative cells, the last vestiges of organelle nuclei in vegetative cells did not disappear completely. They remained in evidence in the vegetative cells during germination of the pollen tubes. InP. zonale, however, no evidence of degradation of organelle nuclei was ever observed. As a result, a very large number of organelle nuclei remained in the sperm cells during maturation of the pollen grains. When the total DNA isolated from the pollen or pollen tubes was analyzed by Southern hybridization with a probe that contained therbc L gene, for detection of the plastid DNA and a probe that contained thecox I gene, for detection of the mitochondrial DNA, the same results were obtained. Therefore, the maternal inheritance of the organelle genes inL. longiflorum is caused by the degradation of the organelle DNA in the generative cells while the biparental inheritance of the organelle genes inP. zonale is the result of the preservation of the organelle DNA in the generative and sperm cells. To characterize the degradation of the organelle nuclei, nucleolytic activities in mature pollen were analyzed by an in situ assay on an SDS-DNA-gel after electrophoresis. The results revealed that a 40kDa Ca²⁺-dependent nuclease and a 23 kDa Zn²⁺ -dependent nuclease were present specifically among the pollen proteins ofL. longiflorum. By contrast, no nucleolytic activity was detected in a similar analysis of pollen proteins ofP. zonale.     

Histone H4 acetylation and DNA methylation dynamics during pollen development

Summary The first pollen mitosis results in generative and vegetative cells which are characterised by a striking difference in their chromatin structure. In this study, histone H4 acetylation and DNA methylation have been analysed during pollen development inLilium longiflorum. Indirect immunofluorescence procedures followed by epifluorescence and laser scanning microscopy enabled a relative quantification of H4 acetylation and DNA methylation in microspores, immature binucleate pollen, mature pollen, and pollen tubes. The results show that histone H4 of the vegetative nucleus, in spite of its decondensed chromatin structure, is strongly hypoacetylated at lysine positions 5 and 8 in comparison with both the original microspore nucleus and the generative-cell nucleus. These H4 terminal lysines in the vegetative nucleus are, however, progressively acetylated during the following pollen tube growth. The DNA methylation analysis inversely correlates with the histone acetylation data. The vegetative nucleus in mature pollen grains is heavily methylated, but a dramatic nonreplicative demethylation occurs during the pollen tube development. Changes neither in H4 acetylation nor in DNA methylation have been found during development of the generative nucleus. The results obtained indicate that the vegetative nucleus enters the quiescent state (accompanied by DNA hypermethylation and H4 underacetylation) during the maturation of pollen grain which enables pollen grains a long-term survival without external source of nutrients until they reach the stigma.     

Comparison of density of nuclear pores on vegetative and generative nuclei in pollen of Tradescantia

The freeze-etch technique has been used to expose surface views of vegetative and generative nuclear envelopes in both ungerminated and germinated pollen of Tradescantia paludosa. A comparison of the density and total numbers of nuclear pores on the two nuclei indicates that vegetative nuclei have at least twice as many pores as generative nuclei. These findings are compatible with the concept that the vegetative nucleus plays a more active role in pollen tube development than the generative nucleus.     

Changes in volume,surface area,and frequency of nuclear pores on the vegetative nucleus of tobacco pollen in fresh,hydrated, and activated conditions

The vegetative nucleus (VN) of Nicotiana tabacum L. has been qualitatively and quantitatively studied in fresh, hydrated, and activated pollen. Techniques included the use of optical sectioning by confocal scanning laser microscopy to obtain volume and surface area measurements, and stereoscopic pairs; and freeze-etch electron microscopy to estimate the frequency of nuclear pores per m² in the vegetative nucleus. Several morphological changes were observed to occur in pollen grain nuclei during the early processes of tube growth. In freshly dehisced pollen grain, the vegetative and generative nuclei were side by side, but following hydration and activation of the grain, the elongated generative nucleus became partially surrounded by the vegetative nucleus. It was found that during hydration, the surface area of the vegetative nucleus increased and there was a decrease in the frequency of nuclear pores. The calculated total number of pores remained similar. After activation and pollen-tube growth, the vegetative nucleus retained the same surface area as in the hydrated state but the frequency of nuclear pores decreased; therefore, the calculated total number of pores was significantly lowered. When considered alongside complementary biochemical data, these morphological results indicate that RNA production in the vegetative nucleus decreases following germination.Abbreviations VN vegetative nucleus (nuclei) - GN generativenucleus - GC generative cell - CSLM confocal scanning laser microscope We acknowledge research support by the Biotechnology Action Programm of the Commission of European Communities, and CNR for the fellowship awarded to Dr. Wagner. We would also like to thank Mrs. C. Faleri for the expert technical help.     

Division of the generative nucleus in cultured pollen tubes of the Bromeliaceae

Pollen germination, division of the generative nucleus and position of the generative nucleus in the pollen tube during in vitro germination were examined for six bromeliad cultivars. The influence of mixed amino acids (casein hydrolysate) and individual amino acids (Arg, Asn, Asp, Glu, Gly, Met, Phe, Orn, Tyr) were tested. Aechmea fasciata and A. chantinii pollen tubes showed more generative nuclear division in cultured pollen tubes than the other four cultivars tested. Casein hydrolysate did not stimulate generative nuclear division. In general arginine (1 mM) improved division of the Aechmea generative nucleus and to a lesser extent this of Vriesea `Christiane', Guzmania lingulata and Tillandsia cyanea. A concentration of 2 mM arginine reduced pollen tube growth of Aechmea. The vegetative nucleus was ahead of the generative nucleus in approximately 50% of the pollen tubes of all cultivars studied. In about 25% of the pollen tubes, the generative nucleus was ahead and in ±25% pollen tubes the vegetative and generative nuclei were joined together. The distance between the two generative nuclei and the distance from the generative nuclei to the pollen tube tip differed significantly for Aechmea fasciata and A. chantinii. The influence of different amino acids for Aechmea fasciata and A. chantinii varied with respect to pollen germination and generative nuclear division. Arg and Met improved nuclear division of both Aechmea cultivars. Pollen germination and sperm cell production were not linked. This information is important to ameliorate in vitro pollination methods used to overcome fertilization barriers in Bromeliaceae and other higher plants.     

Electron microscopic ammonical silver reaction forTradescantia pollen grain

Summary The postformalin ammonical silver reaction (ASR) for basic nuclear proteins was applied to both the generative and vegetative nuclei ofTradescantia pollen grains and examined with the electron microscope. The ASR deposit was usually observed mainly in the dense chromatic regions of both the generative and vegetative nuclei. Since a large amount of dense chromatic regions was observed in the generative nucleus than the vegetative ones in late or mature pollen grains, as a results, the increased amount of the ASR deposit was revealed in the generative nucleus.     

An autoradiographic study of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger

Summary The pattern of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger was studied using³H-uridine autoradiography. Incorporation of label during pollen maturation was periodic with peak RNA synthesis occurring in the uninucleate, nonvacuolate pollen grains and in the vegetative cell of the bicellular pollen grains. During the early stages of germination, isotope incorporation occurred predominantly in the nucleus of the vegetative cell with little or no incorporation in the generative cell. With the appearance of the pollen tube, incorporation of³H-uridine in the vegetative cell nucleus decreased and completely disappeared at later stages of germination. No incorporation of isotope was observed in the sperms formed in the pollen tube by the division of the generative cell. From a comparison of the results of this study with those of previous works on RNA synthesis during pollen embryogenesis in cultured anthers ofH. niger, it is concluded that in contrast to embryogenic development, there is no requirement for sustained RNA synthesis by the generative cell nucleus for normal gametophytic development.     

Changing patterns of nucleic acids, basic and acidic proteins in generative nuclei during pollen germination and pollen tube growth in Hippeastrum belladona

Generative and vegetative nuclei of mature and germinated pollen grains from Hippeastrum belladonna were separated in a continuous Ficoll gradient. Less than 3% contamination was observed between the generative and vegetative nuclear fractions. The vegetative nuclei were composed of two populations; the larger population consisted of nuclei with 1C levels of DNA and the smaller with 2C levels. The generative nuclei consisted of a homogeneous population composed of nuclei possessing 2C levels of DNA. Histone synthesis did not occur in vegetative nuclei. Changes appeared in the gel-electrophoretic banding patterns of the F1 histones of vegetative nuclei during germination. Changes were not observed in the generative nuclei. A reduction of general proteins and RNA was observed in vegetative nuclei by 20 h of germination. The phenol-soluble nuclear proteins of vegetative nuclei revealed transitions in electrophoretic banding patterns during pollen germination that were greater than those shown by the histones. These changes in the PSNP primarily involved reduced concentrations of certain proteins rather than synthesis of new ones. However, a new band was observed in the electrophoretic pattern of the PSNP of vegetative nuclei after 12 h of pollen tube growth. No transition was seen in the PSNP of generative nuclei during pollen germination and tube growth. The regulatory role of the PSNP in cell differentiation is discussed in the light of these findings.     

Patterns of DNA synthesis during pollen embryogenesis in henbane

Continued DNA synthesis in the generative cell nucleus, followed by mitosis and cytokinesis, results in the formation of pollen embryoids in cultured anthers of H. niger. In contrast, the nucleus of the vegetative cell undergoes no DNA synthesis after it is cut off, or synthesizes DNA only during a limited number of cell cycles. DNA synthetic patterns in the generative and vegetative cell nuclei confirm the ontogeny of embryoids described in this plant.