Male germ line development in Arabidopsis. duo pollen mutants reveal gametophytic regulators of generative cell cycle progression

Male germ line development in flowering plants is initiated with the formation of the generative cell that is the progenitor of the two sperm cells. While structural features of the generative cell are well documented, genetic programs required for generative cell cycle progression are unknown. We describe two novel Arabidopsis (Arabidopsis thaliana) mutants, duo pollen1 (duo1) and duo pollen2 (duo2), in which generative cell division is blocked, resulting in the formation of bicellular pollen grains at anthesis. duo1 and duo2 map to different chromosomes and act gametophytically in a male-specific manner. Both duo mutants progress normally through the first haploid division at pollen mitosis I (PMI) but fail at distinct stages of the generative cell cycle. Mutant generative cells in duo1 pollen fail to enter mitosis at G2-M transition, whereas mutant generative cells in duo2 enter PMII but arrest at prometaphase. In wild-type plants, generative and sperm nuclei enter S phase soon after inception, implying that male gametic cells follow a simple S to M cycle. Mutant generative nuclei in duo1 complete DNA synthesis but bypass PMII and enter an endocycle during pollen maturation. However, mutant generative nuclei in duo2 arrest in prometaphase of PMII with a 2C DNA content. Our results identify two essential gametophytic loci required for progression through different phases of the generative cell cycle, providing the first evidence to our knowledge for genetic regulators of male germ line development in flowering plants.     

Phosphorylation of threonine 161 in plant cyclin-dependent kinase A is required for cell division by activation of its associated kinase

Although A-type cyclin-dependent kinase A (CDKA) is required for plant cell division, our understanding of how CDKA is activated before the onset of commitment to cell division is limited. Here we show that phosphorylation of threonine 161 (T161) in plant CDKA is required for activation of its associated kinase. Western blot analysis revealed that phosphorylation of CDKA T161 increased greatly, in parallel with activation of p13(suc1)-associated kinase activity, when stationary-phase tobacco BY-2 cells were subcultured into fresh medium. Although induced over-expression of a dominant-negative CDKA mutant (D146N) fused with green fluorescent protein (GFP) in BY-2 cells resulted in elongated cells after cell division was arrested, over-expression of this CDKA mutant with a non-phosphorylatable alanine in place of T161 (T161A) had no effect on cellular growth. However, immunoprecipitates of both GFP-fused CDKAs exhibited virtually no histone H1 kinase activity, suggesting that both mutants formed kinase-inactive complexes. In a baculovirus expression system, the recombinant CDKA(T161A)/cyclin D complex possessed no detectable kinase activity, indicating that phosphorylation of T161 is required for CDKA activation. To further elucidate the role of T161 phosphorylation, we used a loss-of-function mutation in the CDKA;1 gene, which encodes the only Arabidopsis CDKA. This mutant displays male gametophyte lethality, and produces bicellular pollen grains instead of the tricellular grains produced in wild-type plants. Introduction of CDKA;1(T161E)-GFP, which mimics phosphorylated T161, resulted in successful complementation of the cdka-1 mutation, whereas no recovery was observed when CDKA;1(T161A)-GFP was introduced. Thus, phosphorylation of T161 in Arabidopsis CDKA;1 is essential for cell division during male gametogenesis.     

Chromatin assembly factor 1 regulates the cell cycle but not cell fate during male gametogenesis in Arabidopsis thaliana

The interdependence of cell cycle control, chromatin remodeling and cell fate determination remains unclear in flowering plants. Pollen development provides an interesting model, as it comprises only two cell types produced by two sequential cell divisions. The first division separates the vegetative cell from the generative cell. The generative cell divides and produces the two sperm cells, transported to the female gametes by the pollen tube produced by the vegetative cell. We show in Arabidopsis thaliana that loss of activity of the Chromatin assembly factor 1 (CAF1) pathway causes delay and arrest of the cell cycle during pollen development. Prevention of the second pollen mitosis generates a fraction of CAF1-deficient pollen grains comprising a vegetative cell and a single sperm cell, which both express correctly cell fate markers. The single sperm is functional and fertilizes indiscriminately either female gamete. Our results thus suggest that pollen cell fate is independent from cell cycle regulation.     

An original mutation in soybean (Glycine max (L.) Merrill) involving degeneration of the generative cell and causing male sterility.

A spontaneous mutation causing male sterility has been detected in line BR97-17739 from the soybean breeding program conducted by Embrapa-National Soybean Research Center. Meiotic division and male gametophyte development were analyzed in 10 male-sterile, female-fertile plants. Meiotic process had few irregularities related to chromosome segregation and affected about 2% of tetrads. Despite the high frequency of normal microspores, pollen sterility was total. After callose dissolution, microspores were released into the anther loculle and interphase nucleus was displaced from the center to one side of the cell. Displacement continued throughout normal microspore mitosis (PMI). After telophase, the hemispherical phragmoplast marked the place of cytokinesis. A typical generative cell, adjacent to the plasma membrane, and the vegetative one, containing most of the cytoplasm, were formed. In spite of the well-formed generative cell, pollen mitosis (PMII) failed to occur. The generative cell degenerated and was completely destroyed. The 3:1 segregation for male sterility in this line and its progenies indicate that a single recessive gene controls mutation.     

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.     

被子植物生殖细胞与精细胞的分离方法

开花植物精细胞的发育经历一个独特的后减数分裂过程,在此过程中每个花粉母细胞减数分裂的产物——小孢子经不对称有丝分裂产生1个大的营养细胞和1个小的生殖细胞,随后生殖细胞经过正常的有丝分裂产生2个精细胞。近几年,随着高通量组学技术的不断完善,利用组学技术比较分析生殖细胞和精细胞的分子特征、揭示决定精细胞命运与功能以及受精识别的重要分子已成为植物生殖生物学备受关注的课题。开展此项研究的关键是建立能获得大量高纯度的生殖细胞与精细胞分离纯化技术。该文综述了被子植物生殖细胞和精细胞分离方法的主要研究进展,分析了关键方法的特点和要点以及不同方法之间的差异和共性,以期为相关领域的研究人员提供借鉴。     

芒果花药发育的细胞化学研究

芒果花药发育中,花药药壁体细胞中淀粉粒多糖和脂滴类物质一直很少,仅药室内壁细胞中有零星淀粉粒分布.到二胞花粉早期,花粉营养细胞中的大液泡消失,开始积累淀粉粒.芒果成熟花粉中储存营养物质主要是淀粉粒,而脂类物质一直很少.     

A novel class of MYB factors controls sperm-cell formation in plants

In contrast to animals, the plant male germline is established after meiosis in distinctive haploid structures, termed pollen grains. The germline arises by a distinct asymmetric division of the meiotic products . The fates of the resulting vegetative and generative cells are distinct. In contrast to the larger vegetative cell, arrested in the G1 phase of the cell cycle, the smaller generative cell divides once to produce the two male gametes or sperm cells. Sperm cells are delivered to the female gametes by the pollen tube, which develops from the vegetative cell. In spite of recent efforts to understand pollen development , the molecular pathway controlling sperm-cell ontogenesis is unknown. Here, we present the isolation of DUO1, a novel R2R3 MYB gene of Arabidopsis, as the first gene shown to control male gamete formation in plants. DUO1 is specifically expressed in the male germline, and DUO1 protein accumulates in sperm-cell nuclei. Mutations in DUO1 produce a single larger diploid sperm cell unable to perform fertilization. DUO1 appears to be evolutionarily conserved in several plant species and defines a new subfamily of pollen-specific MYB genes.     

Ultrastructural studies on plastids of generative and vegetative cells inLiliaceae 3. Plastid distribution during the pollen development inGasteria verrucosa (Mill.) Duval

Summary This paper describes the development of pollen grains ofGasteria verrucosa from the late microspore to the mature two-cellular pollen grain. Ultrastructural changes and the distribution of plastids as a result of the first pollen mitosis have been investigated using light and electron microscopy. The microspores as well as the generative and the vegetative cell contain mitochondria and other cytoplasmic organelles during all of the observed developmental stages. In contrast, the generative cell and the vegetative cell show a different plastid content. Plastids are randomly distributed within the microspores before pollen mitosis. During the prophase of the first pollen mitosis the plastids become clustered at the proximal pole of the microspore. The dividing nucleus of the microspore is located at the distal pole of the microspore. Therefore, the plastids are not equally distributed into both the generative and the vegetative cell. The possible reasons for the polarization of plastids within the microspore are briefly discussed. The lack of plastids in the generative cell causes a maternal inheritance of plastids inGasteria verrucosa.     

Generative Cell Division and Sperm Cell Association in the Pollen Grain of Sambucus nigra

Generative cell division in tricellular pollen grains of Sambucusnigra L. (Caprifoliaceae) has been examined with light and electronmicroscopy. During division the generative cell is located inthe centre of the pollen grain, near to the nucleus of a surroundingvegetative cell. Conventional mitosis of the generative cellis followed by cytokinesis through centrifugal cell plate formation.Two sister sperm cells remain in spatial contact with each otherand are surrounded, as formerly their progenitor cell was, bythe vegetative cell. From the changes of shape of the generativecell during division and of the sperm cells it may be assumedthat the space between these cells and the vegetative one containsa labile, non-rigid, wall material. No plastids have been observedin the generative cell and its mitochondria appear to be unequallydistributed between the two future sperm cells during division. Sambucus nigra L., generative cell division, pollen, sperm cell association     

Cytoplasmic DNA apportionment and plastid differentiation during male gametophyte development in Pelargonium zonale

In the male gametophyte of Pelargonium zonale, generative and sperm cells contain cytoplasmic DNA in high density compared to vegetative cells. Cytoplasmic DNA was examined using the DNA fluorochrome DAPI (4'6-diamidino-2-phenylindole) and observed with epifluorescence and electron microscopy. The microspore cell contains a prominent central vacuole before mitosis; mitochondria and plastids are randomly distributed throughout the cytoplasm. Following the first pollen grain mitosis, neither the vegetative cell nor the early generative cell display a distributional difference in cytoplasmic DNA, nor is there in organelle content at this stage. During the maturation of the male gametophyte, however, a significant discrepancy in plastid abundance develops. Plastids in the generative cell return to proplastids and do not contain large starch grains, while those in the vegetative cell develop starch grains and differentiate into large amyloplasts. Plastid nucleoids in generative and sperm cells in a mature male gametophyte are easily discriminated after DAPI staining due to their compactness, while those in vegetative cells stained only weakly. The utility of the hydrophilic, non-autofluorescent resin Technovit 7100 in observing DAPI fluorescence is also demonstrated.     

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.     

Flow Cytometric Determination of Relative Nuclear DNA Contents in Bicellulate and Tricellulate Pollen

Nuclear DNA content in mature pollen was measured with a flowcytometer Pollen of Lilium longiflorum, Dendranthema grandiflora(syn Chrysanthemum monfolium) and Zea mays was chopped and stainedwith the DNA fluorochrome DAPI DNA levels, expressed as arbitraryC values, were compared with those of nuclei isolated from leafor root material of the same plants In mature tricellulate pollen the generative cell is dividedafter second pollen mitosis into two sperm cells Tricellulatepollen from maize and chrysanthemum gave rise to one large 1Cpeak and, only in the case of chrysanthemum, a much smallerone at the 2C level These results suggest that the haploid nucleiof the vegetative as well as both sperm cells in tricellulatepollen are arrested in the G₁ stage of nuclear division Thesmall 2C peak in the case of chrysanthemum probably arose froma fraction of pollen with the sporophytic chromosome number(2n pollen) In contrast to this, mature bicellulate lily pollengave rise to two identical peaks at the 1C and the 2C levelFrom this result it was concluded that in bicellulate pollen,the 1C peak is caused by the signal of the haploid vegetativenucleus arrested in the G₁ stage of nuclear division, whereasthe 2C peak originates from the haploid generative nucleus whichhas already undergone DNA synthesis and is arrested in G₂ Lilium longiflorumThunb, lily, Dendranthema grandiflora Tzelev (syn Chrysanthemum morifolium Ramat ), chrysanthemum, Zea maysL, maize, male gametophytic cells, vegetative cells, generative cells, sperm cells, unreduced pollen, sporophytic cells, relative nuclear DNA contents, replication stage     

A candidate protein kinase C gene, PKC1, is required for the S. cerevisiae cell cycle

Probes derived from cDNAs encoding isozymes of rat protein kinase C (PKC) were used to screen the genome of the budding yeast S. cerevisiae. A single gene (PKC1) was isolated that encodes a putative protein kinase closely related to the alpha, beta, and gamma subspecies of mammalian PKC. Deletion of PKC1 resulted in recessive lethality. Cells depleted of the PKC1 gene product displayed a uniform phenotype, a characteristic of cell division cycle (cdc) mutants, and arrested cell division at a point subsequent to DNA replication, but prior to mitosis. Unlike most cdc mutants, which continue to grow in the absence of cell division, PKC1-depleted cells arrested growth with small buds. PKC1 may regulate a previously unrecognized checkpoint in the cell cycle.     

Distinct chromatin environment associated with phosphorylated H3S10 histone during pollen mitosis I in orchids

Pollen developmental pathway in plants involving synchronized transferal of cellular divisions from meiosis (microsporogenesis) to mitosis (pollen mitosis I/II) eventually offers a unique “meiosis-mitosis shift” at pollen mitosis I. Since the cell type (haploid microspore) and fate of pollen mitosis I differ from typical mitosis (in meristem cells), it is immensely important to analyze the chromosomal distribution of phosphorylated H3S10 histone during atypical pollen mitosis I to comprehend the role of histone phosphorylation in pollen development. We investigated the chromosomal phosphorylation of H3S10 histone during pollen mitosis I in orchids using immunostaining technique. The chromosomal distribution of H3S10ph during pollen mitosis I revealed differential pattern than that of typical mitosis in plants, however, eventually following the similar trends of mitosis in animals where H3S10 phosphorylation begins in the pericentromeric regions first, later extending to the whole chromosomes, and finally declining at anaphase/early cytokinesis (differentiation of vegetative and generative cells). The study suggests that the chromosomal distribution of H3S10ph during cell division is not universal and can be altered between different cell types encoded for diverse cellular processes. During pollen development, phosphorylation of histone might play a critical role in chromosome condensation events throughout pollen mitosis I in plants.     

The cdc7 protein kinase is a dosage dependent regulator of septum formation in fission yeast.

Mutation of the Schizosaccharomyces pombe cdc7 gene prevents formation of the division septum and cytokinesis. We have cloned the cdc7 gene and show that it encodes a protein kinase which is essential for cell division. In the absence of cdc7 function, spore germination, DNA synthesis and mitosis are unaffected, but cells are unable to initiate formation of the division septum. Overexpression of p120cdc7 causes cell cycle arrest; cells complete mitosis and then undergo multiple rounds of septum formation without cell cleavage. This phenotype, which is similar to that resulting from inactivation of cdc16 protein, requires the kinase activity of p120cdc7. Mutations inactivating the early septation gene, cdc11, suppress the formation of multiple septa and allow cells to proliferate normally. If formation of the division septum is prevented by inactivation of either cdc14 or cdc15, p120cdc7 overproduction does not interfere with other events in the mitotic cell cycle. Septation is not induced by overexpression of p120cdc7 in G2 arrested cells, indicating that it does not bypass the normal dependency of septation upon initiation of mitosis. These findings indicate that the p120cdc7 protein kinase plays a key role in initiation of septum formation and cytokinesis in fission yeast and suggest that p120cdc7 interacts with the cdc11 protein in the control of septation.     

Mitosis and Amitosis of Generative Cell Nuclei in Artificially Germinated Pollen Tubes in Zephyranthes candida(Lindl.)Herb.

This paper deals with the comportmem of the vegetative nucleus and its spatial association with the generative cell and sperm cells in the artificially germinated pollen tubes of Zephyranthes candida (Lindl.) Herb. before and after generative cell mitosis with the use of DNA-specific fluochrome 4′,6-diamidino-2-phenylindole (DAPI). The induction of amitosis and abnormal mitosis of generative cell nuclei by cold-pretreatment of the pollen prior to germination was studied in particular. In normal case, the generative cell, after appressing to the vegetative nucleus for certain time, underwent mitosis to form two sperms, while the vegetative nucleus became markedly elongated, diffused, and exhibited blurring of its fluorescence. After division, a pair of sperms remained shortly in close connexion with the vegetative nucleus. Then the vegetative nucleus returned to its original state. In the pollen tubes germinated from cold-pretreated pollen, amitosis of some generative cell nuclei were frequently observed. Amitosis took place via either equal or unequal division with a mode of constriction. During amitosis, the dynamic change of vegetative nucleus and its intimate association with generative cell afore described did not occur. Sperm nuclei produced from amitosis could farther undergo amitisis resulting in micronnclei. Factors affecting the amitosic rate of generative cells, such as pollen developmental stage, temperature and duration of cold-pretreatment, were studied. Besides amitosis, cold-pretreatment also induced some abnormal mitotic behavior leading to the formation of micronuclei. Based on our observations and previously reported facts in other plant materials, it is inferred that the vegetative nucleus plays an important role in normal mitosis of generative cell and development of sperms.     

青葙花药发育的结构和组织化学观察

对苋科植物青葙Celosia argentea花药发育的结构和组织化学（多糖和脂滴）特征进行观察。青葙小孢子发生为同时型,四分体为四面体型。药壁为典型四层,绒毡层属于同型绒毡层。成熟花粉为二胞型。早期花药中的淀粉粒和脂滴均较少,绒毡层细胞至小孢子晚期退化为体积较大的脂块。二胞花粉时期的中层细胞退化为脂滴。早期二胞花粉中先出现多糖颗粒,晚期的成熟花粉中积累大量淀粉粒和较少的脂滴为营养储存物。