Analysis of gemini pollen 3 mutant suggests a broad function of AUGMIN in microtubule organization during sexual reproduction in Arabidopsis

In flowering plants, male gametes arise via meiosis of diploid pollen mother cells followed by two rounds of mitotic division. Haploid microspores undergo polar nuclear migration and asymmetric division at pollen mitosis I to segregate the male germline, followed by division of the germ cell to generate a pair of sperm cells. We previously reported two gemini pollen (gem) mutants that produced twin‐celled pollen arising from polarity and cytokinesis defects at pollen mitosis I in Arabidopsis. Here, we report an independent mutant, gem3, with a similar division phenotype and severe genetic transmission defects through pollen. Cytological analyses revealed that gem3 disrupts cell division during male meiosis, at pollen mitosis I and during female gametophyte development. We show that gem3 is a hypomorphic allele (aug6‐1) of AUGMIN subunit 6, encoding a conserved component in the augmin complex, which mediates microtubule (MT)‐dependent MT nucleation in acentrosomal cells. We show that MT arrays are disturbed in gem3/aug6‐1 during male meiosis and pollen mitosis I using fluorescent MT‐markers. Our results demonstrate a broad role for the augmin complex in MT organization during sexual reproduction, and highlight gem3/aug6‐1 mutants as a valuable tool for the investigation of augmin‐dependent MT nucleation and dynamics in plant cells.     

凤仙花花药发育中多糖和脂滴组织化学研究

以不同发育时期的凤仙花花药为实验材料,采用组织化学方法,对花药发育中的结构变化及多糖和脂滴物质分布进行观察。结果表明:(1)凤仙花的花药壁由6层细胞组成,包括1层表皮细胞,2层药室内壁细胞,2层中层细胞和1层绒毡层细胞。其中绒毡层细胞的形态不明显,很难与造孢细胞区分,且在小孢子母细胞时期退化。(2)在小孢子母细胞中出现了一些淀粉粒,但减数分裂后,早期小孢子中的淀粉粒消失,又出现了一些小的脂滴;随着花粉的发育,小孢子形成大液泡,晚期小孢子中的脂滴也消失;小孢子分裂形成二胞花粉后,营养细胞中的大液泡降解、消失,二胞花粉中又开始积累淀粉;接近开花时,成熟花粉中充满细胞质,其中包含了较多的淀粉粒和脂滴。(3)在凤仙花的花药发育中,绒毡层细胞很早退化,为小孢子母细胞和四分体小孢子提供了营养物质;其后的中层细胞退化则为后期花粉发育提供了营养物质。     

Pro-embryos induction from <Emphasis Type="Italic">Olea europaea</Emphasis> L. isolated microspore culture

Studies were undertaken with one olive (Olea europaea L.) cultivar to identify buds with microspores competent to embryogenesis in vitro. Isolated microspore cultures were performed for the induction of gametic embryogenesis. Different pollen development stages and stress conditions (heat or cold shock) were evaluated. The correlation of inflorescence, anther morphology and the suitable stage of microspore development were analysed. The morphology of responsive buds was identified which corresponded with microspores from the late uni-nucleate to early bi-nucleate pollen stages. Symmetrical divisions of microspores as well as resulting multinucleate structures and pro-embryos were observed. In this paper, a new method of isolated microspore culture that leads to cell division and pro-embryos in olive, is reported.     

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.     

Manipulation of division symmetry and developmental fate in cultures of potato microspores

The effect of media composition on microspore culture was investigated in one tetraploid and two diploid potatoes. The viability of microspores isolated from 4.5 to 5 mm buds was in the range of 33 to 52%. In media for anther culture, microspores showed no further development and lost viability within 2 days. In M1 medium containing mineral components, sucrose, uridine, cytidine, myo-inositol, glutamine and lactalbumin hydrolysate, 18 to 37% of microspores underwent mitosis within 14 days. Up to 95% of the divisions were symmetric and produced equal nuclei. Some symmetrically divided microspores eventually produced structures with 3 to 10 nuclei. The proportion of the total microspore population producing multinuclear structures reached 9% in diploid clones responsive to anther culture and 1 to 2% in recalcitrant cv. Borka. Symmetric mitoses in M1 medium were induced in the presence of glutamine and lactalbumin hydrolysate. Nucleosides and myo-inositol had no effect on microspore division. In the absence of all organic components except sucrose, most mitoses were asymmetric, formation of multinuclear structures was reduced and most pollen accumulated starch indicative of gametophytic fate. In complete M1 medium, starch accumulation was suppressed. Suppression also occurred in asymmetrically divided microspores, indicating a direct inhibition of pollen development independent of the mode of microspore division. This inhibitory effect of M1 medium might present a stress which triggers the induction of symmetric microspore division and subsequent formation of multinuclear structures. This revised version was published online in June 2006 with corrections to the Cover Date.     

An investigation of the role of the anther tapetum during microspore development using genetic cell ablation

The effects on anther development of a fusion of the Arabidopsis anther-specific apg gene promoter to a ribonuclease (barnase) in transgenic tobacco plants were examined. Contrary to expectations, viable pollen grains were produced by these plants despite the demonstration that ribonuclease expression in the microspores and tapetum caused targeted cell ablation. Transformed plants were reduced in male fertility due to ablation of a proportion of pollen dependent on apg-barnase locus number. Plants were otherwise phenotypically normal and fully female fertile, confirming the anther-specific nature of the apg promoter. In microspores inheriting an apg-barnase locus following meiosis, loss of cell viability, as judged by fluorescein diacetate staining, occurred during mid to late microspore development. Microspores not inheriting a transgene went on to mature into viable pollen grains. Premature degeneration of the tapetum was also observed as a result of apg-barnase expression, but this did not appear to disrupt the subsequent microspore and pollen developmental programmes. This was substantiated by observations of microspore development in plants in which the tapetum was rescued from ablation by crossing in a second transgene encoding a tapetum-specific inhibitor of the ribonuclease. It was determined that tapetum cell disruption occurs at the early to mid uninucleate microspore stage in apg-barnase transformants. The data presented show that after this point in microspore development the tapetum is no longer essential for the production of viable pollen in tobacco.     

Scanning electron microscopy of microspore embryogenesis inBrassica spp.

Scanning electron microscopy was employed to study and compare microspore embryogenesis in vitro with pollen development in planta inBrassica napus andB. oleracea. An exine with its specific pattern had already been formed, when microspores were released from tetrads. During subsequent pollen development, microspores increased in size and continued to strengthen the exine. Upon in vitro culture, all microspores, i.e., embryogenic and nonembryogenic, initially showed the same morphological features. After 24 h in culture, the microspores had increased in size. Thereafter, embryogenesis was indicated in some microspores by two different morphological changes. One featured an expansion in volume of the cell cluster around the germination aperture (type I), the other showed cell cluster volume expansion over the entire microspore surface (type II). Two-thirds of embryogenic microspores in bothB. napus andB. oleracea demonstrated type I development. When followed by fluorescence microscopy, in vitro culture of microspores revealed cultures with a high embryo frequency were those with a high frequency of symmetrical division.Abbreviations SEM Scanning electron microscopy - TEM Transmission electron microscopy     

SIDECAR POLLEN suggests a plant-specific regulatory network underlying asymmetric microspore division in Arabidopsis

Asymmetric cell division is a universal strategy to generate diverse cell types necessary for patterning and proliferation of all eukaryotes. The development of haploid male gametophytes (pollen grains) in flowering plants is a remarkable example in which division asymmetry governs the functional specialization and germline differentiation essential for double fertilization. The male gametophyte is patterned via two mitotic divisions resulting in three highly differentiated daughter cells at maturity, a vegetative cell and two sperm cells. The first asymmetric division segregates a unique male germ cell from an undetermined haploid microspore and is executed in an elaborate sequence of cellular events. However the molecular mechanisms governing the division asymmetry in microspores are poorly understood. Recently we studied the phenotype of sidecar pollen (scp) mutants in detail, and demonstrated a requirement of SCP for both the correct timing and orientation of microspore division. SCP is a microspore-specific member of the LOB/AS2 domain family (LBD27/ASL29) showing that a plant-specific regulator plays a key role in oriented division of polarized microspores. Identification of SCP will serve as a new platform to further explore the largely unknown molecular networks regulating division asymmetry in microspores that establishes the male germline in flowering plants.Key words: sidecar pollen, microspore division, division asymmetry, male gametophyte development, male germline, LBD/ASL family proteinUnlike animals, flowering plants do not set aside a distinct germline from an early stage of the life cycle. Instead the angiosperm germline or germ cells are only segregated in the male and female gametophytes by a limited number of post-meiotic mitoses.¹ However, in common with their metazoan cousins, angiosperms utilize division asymmetry for cellular patterning and differentiation of their germlines. Through the unique patterning of a ‘cell-within-a-cell’ structure with three highly differentiated cells, the male gametophyte (pollen grains) serves its biological role to deliver two sessile male gametes to the female gametophyte. Two sequential but different modes of mitotic divisions pattern the male gametophyte (Fig. 1).² The first division (of the microspore) is asymmetric giving rise to two completely different daughter cells, a larger vegetative cell that will form the pollen tube and a smaller germ cell that is engulfed within the vegetative cell cytoplasm. The second division (of the germ cell) usually appears symmetric^† and produces a pair of linked sperm cells. Microspores artificially induced to undergo symmetric division using microtubule inhibitors lack the germ cell and fail to form the typical three-celled structure showing that asymmetry in microspore division is critical for patterning of the male gametophyte.⁴Open in a separate windowFigure 1Male gametophyte development in Arabidopsis (upper part) and mutations that block germ cell formation (lower part). (Upper part) Male gametophyte development involves two rounds of mitotic division. Prior to the first division the centrally positioned microspore nucleus migrates towards the radial wall (the future germ cell pole marked with an asterisk). At this eccentric site the polarized microspores undergo oriented mitosis and cytokinesis giving rise to highly unequal daughter cells, a vegetative cell and a germ cell of which the later produces a pair of sperm cells by symmetric division. (Lower part) Mutants that fail to establish a distinct germ cell arising from specific defects are illustrated. Arrows in red indicate the developmental origin of the phenotypic defects in mutants. Note that two daughter nuclei in the mutants are in grey to show that their cell fates have not yet been thoroughly investigated. n, nucleus; Vn, vegetative nucleus; Gn, generative nucleus; Gc, generative (or germ) cell; Sc, sperm cell; WT, wild type; gem1, gemini pollen1; scp, sidecar pollen; tio, two-in-one; hik/tes, hinkel/tetraspore 12a/12b, kinesin-12a/kinesin-12b.     

含笑花药发育的超微结构研究

对含笑花药发育中的超微结构变化进行观察,结果显示:(1)花粉发育中有三次液泡变化过程——第一次是小孢子母细胞在形成时内部出现了液泡,这可能与胼胝质壁的形成有关;第二次是在小孢子母细胞减数分裂之前,细胞内壁纤维素降解区域形成液泡,它的功能可能是消化原有的纤维素细胞壁;第三次是在小孢子液泡化时期,形成的大液泡将细胞核挤到边缘,产生极性。(2)含笑花粉在小孢子早期形成花粉外壁外层,花粉外壁内层在小孢子晚期形成,而花粉内壁是在二胞花粉早期形成;花粉成熟时,表面上沉积了绒毡层细胞的降解物而形成了花粉覆盖物。研究认为,含笑花粉原外壁的形成可能与母细胞胼胝质壁有关,而由绒毡层细胞提供的孢粉素物质按一定结构建成了花粉覆盖物。     

LP28, a lily pollen-specific LEA-like protein, is located in the callosic cell wall during male gametogenesis

. LP28, a pollen-specific LEA-like protein identified in Lilium longiflorum purportedly related to the desiccation tolerance of pollen, was localized during male gametogenesis using immuno-electron microscopy. At premeiotic interphase, LP28 label is absent from the microsporocyte. LP28 label was first detected in the cell wall of the microsporocyte at meiotic prophase I. LP28 gradually increased as the cell wall thickened. In the dyad, after the first meiotic division, LP28 label also appeared in the septum. In the tetrad, after the second meiotic division, LP28 was detected throughout the cell wall, including the septa. Immunolabeling of callose during meiosis indicated that the appearance and localization of LP28 was very similar to that of callose. After the microspores were released from the tetrad by digesting the callosic cell wall, LP28 was not found in the microspores. In bicellular pollen, just after microspore mitosis, LP28 appeared in the generative cell wall, which also consisted of callose. After pollen germination, LP28 also accumulated in the callosic layer of the elongated pollen tube wall and the callose plug. Thus, LP28 colocalized with the callosic cell wall during male gametogenesis. The possible role of LP28 with respect to wall formation during meiosis and pollen development is discussed.     

罗汉松雄球花及其雄配子体发育的形态结构

该研究采用光学显微镜和扫描电镜,观察罗汉松雄球花、小孢子及其配子体发育过程的形态结构特征,以揭示罗汉松小孢子的发生和雄配子体的发育规律,为罗汉松的生殖和杂交组合提供胚胎学证据。结果发现：(1)罗汉松花芽于每年的7月开始分化,至次年5月花粉成熟散粉,雄球花由单生的卵圆形转为2～3个葇荑花序并生,小孢子叶螺旋状着生于圆柱状的花序轴上,每一小孢子叶远轴面基部并列着生2个小孢子囊。(2)小孢子囊壁发育过程中由外及里出现各由1层薄壁细胞组成的表皮、药室内壁、中层和绒毡层,至散粉前,后两者基本被分解吸收。(3)同一小孢子囊内的造孢细胞发育在时间上存在差异,小孢子母细胞减数分裂后形成的四分体有四面体型和十字交叉型两种排列方式,成熟的雄配子体包括生殖细胞和粉管细胞,发育过程中出现的第一和第二原叶细胞大部分被分解消失。(4)电镜下罗汉松花粉粒为典型的松花型花粉,两侧各具1个气囊,远极面具一萌发沟,花粉粒表面具纹理或皱褶。