Generative Cell Nucleus Research Articles

Proteomic Analysis of Generative and Vegetative Nuclei Reveals Molecular Characteristics of Pollen Cell Differentiation in Lily.

In plants, the cell fates of a vegetative cell (VC) and generative cell (GC) are determined after the asymmetric division of the haploid microspore. The VC exits the cell cycle and grows a pollen tube, while the GC undergoes further mitosis to produce two sperm cells for double fertilization. However, our understanding of the mechanisms underlying their fate differentiation remains limited. One major advantage of the nuclear proteome analysis is that it is the only method currently able to uncover the systemic differences between VC and GC due to GC being engulfed within the cytoplasm of VC, limiting the use of transcriptome. Here, we obtained pure preparations of the vegetative cell nuclei (VNs) and generative cell nuclei (GNs) from germinating lily pollens. Utilizing these high-purity VNs and GNs, we compared the differential nucleoproteins between them using state-of-the-art quantitative proteomic techniques. We identified 720 different amount proteins (DAPs) and grouped the results in 11 fate differentiation categories. Among them, we identified 29 transcription factors (TFs) and 10 cell fate determinants. Significant differences were found in the molecular activities of vegetative and reproductive nuclei. The TFs in VN mainly participate in pollen tube development. In comparison, the TFs in GN are mainly involved in cell differentiation and male gametogenesis. The identified novel TFs may play an important role in cell fate differentiation. Our data also indicate differences in nuclear pore complexes and epigenetic modifications: more nucleoporins synthesized in VN; more histone variants and chaperones; and structural maintenance of chromosome (SMC) proteins, chromatin remodelers, and DNA methylation-related proteins expressed in GN. The VC has active macromolecular metabolism and mRNA processing, while GC has active nucleic acid metabolism and translation. Moreover, the members of unfolded protein response (UPR) and programmed cell death accumulate in VN, and DNA damage repair is active in GN. Differences in the stress response of DAPs in VN vs. GN were also found. This study provides a further understanding of pollen cell differentiation mechanisms and also a sound basis for future studies of the molecular mechanisms behind cell fate differentiation.

Anomalous pollen grain development after nondifferentiating microspore division in Eremurus

Anomalous pollen grain development in <i>Eremurus</i> is caused by an anomalous position of the mitotic spindle and microspore protoplast division into two cells different than in differentiating division. The nuclei of the abnormal gametophytes are always spherical and that of the smaller of the two cells, notwithstanding the shape and position of the latter, has a more compact structure resembling rather that of the generative cell nucleus. Binucleate abnormal gametophytes always have equal nuclei. The wall separating the cells in abnormal pollen grains at first contains callose and, after disappearance of the latter, probably pectins and cellulose. Abnormal pollen grains contain less cytoplasmic RNA than normal ones arid most of them degenerate. If their viability is preserved they do not form' normal pollen tubes in vitro. The frequency of anomalous microspore division is higher in <i>E. robustus</i> (max ca. 50%) than in <i>E. himalaicus</i> (max. ca. 30%) and shows considerable seasonal variations. The results obtained suggest that disturbances in microspore development in <i>Eremurus</i> have a genetic background, but are stimulated by temperature variations in the period preceding mitosis in the microspore.

Detection of differentially expressed variant histone H3.3 in the vegetative nucleus of lily pollen

Histone H3.3, a gene that is differentially expressed in the vegetative cell of lily pollen, was characterized immunologically. Antibody raised against a specific peptide fragment clearly recognized histone H3.3 in mature pollen, and did not cross-react with somatic histone H3s. Immunoblot analysis indicated that the variant histone H3.3 increased during the development of late-bicellular pollen to mature pollen. Immunofluorescence labeling of pollen grains using the specific antibody revealed the presence of variant histone H3.3 only in the vegetative cell nucleus and not in the generative cell nucleus. This is the first study to demonstrate a differentially expressed variant histone H3.3 present in the vegetative cell nucleus. Since the chromatin of the vegetative cell nucleus is largely diffuse and transcriptionally active, the accumulation of a specialized histone H3.3 in the vegetative cell nucleus may potentially control condensation of chromatin and regulate the upregulation of pollen-enhanced genes expressed in the course of pollen germination and tube growth.

Histone H4 acetylation and DNA methylation dynamics during pollen development

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.

Male gametic cell-specific expression of H2A and H3 histone genes.

Formation of the generative cell in flowering plants initiates the male spermatogenesis pathway which eventually culminates in the process of double fertilization. Little is known about the molecular mechanisms operative in the generative cell. Here, we report the isolation and characterization of cDNA clones encoding generative cell-specific histones, gcH2A and gcH3. In situ hybridization analysis revealed that both genes are activated after formation of the generative cell and accumulation of transcripts increases progressively during generative cell maturation. These results suggest that gcH2A and gcH3 mRNAs are products of transcriptional activation of the generative cell nucleus and thus provide the first evidence for male germ line cell-specific gene expression in flowering plants.

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.

Nuclear DNA amount during sporogenesis and gametogenesis in sexual and aposporous buffelgrass

Nuclear DNA amounts (C values) were measured in Feulgen-stained sections of anthers and ovules of sexual plant B-2s (genotype aaaa) and aposporous cultivar Higgins (genotype AAaa) of buffelgrass (Pennisetum ciliare). The mass of the unreplicated nuclear genome of a gamete equals 1C DNA. In both lines, pollen mother cell nuclei were 4C before leptotene; anther wall, dyad, 1-nucleate pollen, and generative cell nuclei were 2C; microspore tetrad, enlarging microspore, and sperm nuclei were 1C. The tapetum persisted as uninucleate cells with 4C DNA. Archespores (2-4C) of both lines initiated meiosis to form megaspore tetrad nuclei with 1-2C DNA. In B-2s, chalazal megaspores (2-4C) formed reduced 8-nucleate Polygonum type embryo sacs, and sacs at 2- and 4-nucleate stages showed distributions with peaks near C1 and C2, corresponding to G1 and G2 cell cycle phases; this is characteristic of active mitosis. Nuclei of 8-nucleate sacs and of eggs and polars were 1C, indicating chromosomes were not duplicated before fertilization. Antipodal nuclei had levels from 1 to 36C, possibly due to polyteny or endopolyploidy. In Higgins, aposporous initials and 2-nucleate embryo sacs showed bimodal distributions of 2n nuclei with peaks at 2C and 4C DNA. Nuclei of newly formed 4-nucleate Panicum type aposporous sacs and of polars were 2C; aposporous eggs stained too faintly for reliable measurement.

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.

RNA synthesis by vegetative and sperm nuclei of trinucleate pollen.

In binucleate pollen tubes the vegetative and generative cell nuclei are known to synthesize RNA. Nothing is known about RNA synthesis in trinucleate pollens. To determine whether RNA synthesis occurs in trinucleate pollen, Secale cereale pollen was studied. The vegetative and sperm cell nuclei of the in vitro grown pollen tubes were shown by autoradiography to incorporate [5-3H] uridine. This incorporation was interpreted as indicating the synthesis of RNA.

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

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.

Single fertilization inSpiranthes sinensis

The reproduction system inSpiranthes sinensis (Orchidaceae), collected at various localities in Japan, was revealed to be of a peculiar new type which is not to be found in other angiosperms. None of generative cell nuclei has been observed to dividede novo throughout the progamic phase, although they do participate in fertilization. Neither fertilization of the generative cell nucleus with the central nucleus nor formation of endosperm occurred in this plant. Although the chromosome number in the developing proembryonic cells numbered 2n=30, exhibiting diploidy, the number of marker chromosomes was equal to the sum of half the number of marker chromosomes of the parental clones.From the results described above, we may conclude that the reproduction system of this species represents a new type of single fertilization (non-double fertilization) between egg cell and sperm cell nuclei caused by the omission of generative cell division and the formation of only one sperm cell nucleus.

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.