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.     

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.     

The basic proteins of male gametic nuclei isolated from pollen grains of Lilium longiflorum

A method has been developed for the efficient isolation of generative and vegetative nuclei from the generative and vegetative cells, respectively, of pollen grains of Lilium longiflorum Thunb. First, large numbers of pollen protoplasts were isolated enzymatically from nearly mature pollen grains. After the protoplasts had been gently disrupted by a mechanical method, the generative cells could be separated from the other pollen contents, which included vegetative nuclei. The generative nuclei were isolated by suspending the purified generative cells in a buffer that contained a non-ionic deter gent. The isolated generative nuclei, like those within pollen grains, had highly condensed chromatin and the isolated material was without contamination by vegetative nuclei. When basic proteins, extracted from the preparation of generative nuclei by treatment with 0.4 N H₂SO₄, were compared with those from preparations of somatic and vegetative nuclei by two-dimensional gel electrophoresis, it was revealed that at least five proteins with apparent molecular masses of 35, 33, 22.5, 21 and 18.5 kDa (p35, p33, p22.5, p21 and p18.5), respectively, were specific for, or highly concentrated in, the generative nuclei. An examination of solubility in 5% perchloric acid and the mobility during electrophoresis indicated that two of these proteins (p35 and p33) resembled H1 histones while the three other proteins (p22.5, p21 and p18.5) resembled core histones. It is likely that these basic nuclear proteins are related to the condensation of chromatin or to the differentiation of male gametes in flowering plants, as is the case for analogous proteins present during spermatogenesis in animals.Abbreviations DAPI 4'6-diamidino-2-phenylindole - NIB nuclear isolation buffer This work was supported in part by Grant-inAid for Scientific Research from the Ministry of Education, Science and Culture, Japan.     

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.     

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.     

ULTRASTRUCTURE OF ANOMALOUS POLLEN DEVELOPMENT IN EMBRYOGENIC ANTHER CULTURES OF HYOSCYAMUS NIGER

The formation of anomalous, binucleate pollen grains and their subsequent embryogenic development, induced by anther culture in Hyoscyamus niger, were analyzed by transmission electron microscopy (TEM). In culture, uninucleate pollen grains occasionally divided symmetrically giving rise to two apparently identical nuclei sharing a common cytoplasm. These nuclei divided once or twice unaccompanied by cell wall formation. After the daughter nuclei organized into cells, their subsequent division products contributed to embryoid formation. In conjunction with previous studies of pollen embryogenesis in H. niger, it appears that in contrast to the principle mode of embryogenesis (i.e., first asymmetric division forms typical two-celled pollen grain and the generative cell acts as the embryogenic precursor), anomalous pollen show no carry-over of gametophytic influences following embryogenic induction. This suggests that specific pathways of embryogenesis are correlated with the rate at which gametophytic gene activity is repressed following induction.     

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     

Isolation and separation of S-competent and S-incompetent nuclei from Tradescantia pollen grains

A method is described, using discontinuous sucrose density gradients, for the separation of generative and vegetative nuclei from young Tradescantia pollen grains during the period of DNA synthesis. The nuclei are obtained in 20–30% yield from the pollen grains; they are 85–95% pure (generative) and 70–80% pure (vegetative).     

Derepression of the cell cycle by starvation is involved in the induction of tobacco pollen embryogenesis

Summary Microspectrophotometry following Feulgen staining and autoradiography following (³H)-thymidine labelling were used to study cell-cycle events during pollen development in tobacco (Nicotiana tabacum L.). During normal gametophytic pollen development in the anther and in vitro the generative nucleus passes through the S phase to the G₂ phase soon after microspore mitosis, while the vegetative nucleus remains arrested in G₁ (=G₀). During embryogenie induction by an in vitro starvation treatment of immature pollen ongoing DNA replication in the generative nucleus is completed and followed by DNA replication in the vegetative cell in a large fraction of the pollen grains. Addition of the DNA replication inhibitor hydroxyurea to the starvation medium postpones S phase entry until the pollen is transferred to a rich medium and does not affect embryo formation. These results demonstrate that one of the crucial events of embryogenic induction is the derepression of the G₁ arrest in the cell cycle of the vegetative cell.     

The isolation and culture of lily pollen protoplasts

Methods for the enzymatic isolation of lily protoplasts and their successful culture are described. When pre-anthesis binucleate pollen (immature pollen grains) was treated in enzyme solution containing macerozyme and cellulase, up to 80% lost their exine and gave rise to intact protoplasts within 1 h. These pollen protoplasts were uniform in size and densely cytoplasmic with two prominent generative and vegetative nuclei. The isolated pollen protoplasts regenerated a cell wall within 1 day of culture and produced a structure resembling a pollen tube after 10–12 days of culture. During this culture period, dividing generative nuclei or 2 sperm nuclei were observed in many protoplasts with regenerated cell walls.     

Generative cells ofLilium longiflorum possess translatable mRNA and functional protein synthesis machinery

Metabolic labelling with [³⁵S]-methionine demonstrated that generative cells ofLilium longiflorum possess their own set of mRNA and are capable of synthesising proteins independently from the vegetative cell. The isolated generative cells synthesised ten proteins, of which six were unique to these specialised cells. Isolation of generative cells from pollen grains after [³⁵S]-methionine labelling resulted in an identical protein profile, therefore the synthesis of these proteins was not due to isolation shock. Addition of cycloheximide, abolished TCA-precipitable counts, whilst actinomycin D had no qualitative effect on the observed protein profile, indicating active translation of pre-existing mRNAs by the generative cells.     

Microgametophytic plastid nucleoid content and reproductive and life history traits of tribeTrifolieae (Fabaceae)

Microgametophytic plastid nucleoids were quantified for 18 species representing the four core genera of the tribeTrifolieae (Fabaceae),Medicago, Melilotus, Trigonella, andTrifolium. Generative cells of all taxa contained nucleoids, establishing that biparental plastid inheritance is common in theTrifolieae. Nucleoid number and volumes of pollen grains and generative cell nuclei differed among taxa. Nucleoid number was positively correlated with pollen grain and generative cell nuclear volumes, flower size and style length. These relationships disappeared after adjusting nucleoid number for pollen grain and generative cell nuclear volumes. Adjusted nucleoid numbers provided no evidence to support hypotheses that plastid content is associated with ploidy level, mating system, perenniality or size of the reproductive apparatus.     

Microtubule organization of in situ and isolated generative cells in Zephyranthes grandiflora Lindl

Summary Microtubule organization in the generative cells of Zephyranthes grandiflora was investigated by immunofluorescence microscopy with a monoclonal anti--tubulin. The experimental materials used were generative cells located within pollen grains and tubes (i.e., in situ) as well as those artificially isolated after osmotic shock or grinding treatments of the pollen grains. Diverse microtubule organization patterns were revealed. In situ, the generative cells appeared spindle-shaped and contained mainly longitudinally oriented microtubule bundles, although other types were found as well. After isolation, as the alteration in microtubule patterns took place, the spindle-shaped generative cells became ellipsoidal and then spherical. In the ellipsoidal cells a transitional form consisting of a mixture of microtubule bundles and meshes could be found. In spherical cells the mesh structure appeared to be the predominant pattern. These results indicate that the microtubule cytoskeleton of the generative cells can change easily from one structural form to another in accordance with environmental conditions and may play an important role in determining the cell shape.     

Male gametic nucleus-specific H2B and H3 histones,designated gH2B and gH3, in Lilium longiflorum

Two proteins that resemble core histones and might be specific to the male gametic (generative) nucleus within the pollen of Lilium longiflorum Thumb, (originally designated p22.5 and p18.5; K. Ueda and I. Tanaka, 1994, Planta, 192, 446–452) were characterized biochemically and immunochemically. Patterns of digestion of p22.5 and p18.5 by Staphylococcus aureus V8 protease closely resembled those of somatic histones H2B and H3, respectively. However, peptide fragments that were unique to p22.5 or p18.5 were also detected. Antibodies raised against these proteins did not cross-react with any somatic histones. These results indicate that p22.5 and p18.5 are different from somatic histones in terms of primary structure. Analysis of their amino-acid compositions revealed that p22.5 is a moderately lysine-rich protein while p18.5 is an arginine-rich protein. From these results, we conclude that p22.5 is a variant of histone H2B and p18.5 is a variant of histone H3. Immunofluorescence staining of pollen grains using the specific antibodies revealed that both p22.5 and p18.5 are only present in the generative cell nucleus and are not to be found in the vegetative cell nucleus. This study demonstrates that (i) specific histone variants are present in the male gametic nucleus of a higher plant, as they are in the sperm nucleus of animals, and (ii) distinct differences in histone composition exist between the nuclei of generative and vegetative cells in pollen. These novel histones (p22.5 and p18.5), specific to male gametic nuclei, have been designated gH2B and gH3, respectively.Abbreviations DAPI 46-diamidino-2-phenylindole - FITC fluorescein isothiocyanate The authors thank Dr. Y. Sado (Shigei Medical Institute, Japan) for his helpful advice on immunization and Prof. T. Iguchi and Prof. K. Manabe (Yokohama City University, Japan) for providing facilities for experiments. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.     

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.     

Isolation of generative cells and their protoplasts from pollen ofLilium longiflorum

Summary Methods are described for the isolation of large quantities of generative cells and their protoplasts from the pollen ofLilium longiflorum. First, large numbers of pollen protoplasts were enzymatically isolated from immature pollen grains. When they were gently disrupted mechanically, the pollen contents including spindle-shaped generative cells were released. The generative cells were separated from other structures by Percoll density gradient centrifugation. They were nearly spherical, but had a callosic cell wall. The isolated generative cells were then re-treated in enzyme solution to yield authentic protoplasts. The generative cell protoplasts, gametoplasts, were uniform in size and contained a condensed haploid nucleus with relatively little cytoplasm.