Cytological mechanism of pollen abortion resulting from allelic interaction of F1 pollen sterility locus in rice (Oryza sativa L.)

Pollen abortion is one of the major reasons causing the inter-subspecific F₁ hybrid sterility in rice and is due to allelic interaction of F₁ pollen sterility genes. The microsporogenesis and microgametogenesis of Taichung 65 and its three F₁ hybrids were comparatively studied by using techniques of differential interference contrast microscopy, semi-thin section light microscopy, epifluorescence microscopy and TEM. The results showed that there were differences among the cytological mechanisms of pollen abortion due to allelic interaction at the three F₁ pollen sterility loci. The allelic interaction at S-a locus resulted in microspores unable to extend the protoplasm membrane with the enlargement of the microspore at the middle microspore stage and finally producing empty abortive pollen. The allelic interaction at S-b locus caused asynchronous development of microspores at the middle microspore stage producing stainable abortive pollen. The allelic interaction at S-c locus mainly led to the non-dissolution of the generative cell wall and finally caused the hybrid F₁ mainly producing stainable abortive pollen. Genotypic identification indicated that the abortive pollen were those with S ^j allele.     

Nuclear pore dynamics during pollen development and androgenesis in Brassica napus

Changes in nuclear pore complex (NPC) densities, NPCs/nucleus and NPCs/μm³, are described using freeze-fractured Brassica napus microspores and pollen in vivo and in vitro. Early stages of microspore- and pollen-derived embryogenic cells were also analysed. The results of in vivo and in vitro pollen development indicate an increase in activity of the vegetative nucleus during maturation of the pollen. At the onset of microspore and pollen culture, NPC density decreased from 15 NPCs/μm² at the stage of isolation to 9 NPCs/μm², under both embryogenic and non-embryogenic conditions. This implies that the drop in NPC density might be a result of culturing the microspores and pollen rather than an indication for microspore and pollen embryogenesis in Brassica napus. However, after 1 day in culture under embryogenic conditions, the NPC density increased again and stabilised around 13 NPCs/μm², whereas under non-embryogenic conditions the NPC density remained about 9 NPCs/μm². This low density of 9 NPCs/μm² was also found in the nuclei of sperm cells, in contrast to the 19 NPCs/μm² found in the vegetative nucleus. It means that, although both the vegetative and sperm nuclei are believed to be metabolically rather inactive in mature pollen, the NPC density of vegetative nucleus is twice as high as the NPC density of the sperm nuclei. In a few cases, embryos formed suspensor-like structures with a NPC density of 9 NPCs/μm², indicating a lower nucleocytoplasmic exchange of the nuclei of the suspensor cells than with the nuclei in the embryo proper. In addition, observations on NPCs and other organelles, obtained by high resolution cryo-scanning microscopy, are presented. Received: 29 December 1999 / Revision accepted: 3 March 2000     

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。     

Substrate specificity of the epoxidation reaction in sex pheromone biosynthesis of the Japanese giant looper (Lepidoptera: Geometridae)

Female moths of the Japanese giant looper (Ascotis selenaria cretacea, Lepidoptera: Geometridae) secrete (Z,Z)-6,9-cis-3,4-epoxynonadecadiene as a sex pheromone component. To the pheromone glands of the decapitated females, [19,19,19-D₃](Z,Z,Z)-3,6,9-nonadecatriene was applied after an injection of pheromone biosynthesis activating neuropeptide. GC-MS analysis of the gland extract showed its specific conversion into the pheromonal cis-3,4-epoxide indicating that the C₁₉ triene which had been identified in the gland was a precursor of the pheromone. In order to examine the substrate specificity of the enzyme catalyzing this epoxidation step, several unsaturated hydrocarbons not occurring in the gland were applied to it. Not only (Z,Z,Z)-3,6,9-trienes with varying chain lengths (C₁₇, C₁₈ and C₂₀ to C₂₂) but (Z,Z)-3,6-dienes (C₁₇, C₁₉ and C₂₀) were converted into the corresponding cis-3,4-epoxides in a rather good yield, while no 6,7- and 9,10-epoxides could be detected. (Z)-3-Nonadecene was also changed to the cis-epoxide, but (E)-3-, (Z)-2- and (Z)-4-double bonds in the C₁₉ chain were not oxidized. These in vivo experiments revealed that the monooxygenase regiospecifically attacked the (Z)-3-double bond of straight chain hydrocarbons regardless of their length and degree of unsaturation.     

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.     

Nucleic acids in relation to cell division in Lilium longiflorum

Methods are described for preparing cell suspensions of Lilium microsporocytes, microspores and pollen grains; for obtaining cell counts of these suspensions; and for their analysis for pentose nucleic acid (PNA) and desoxypentose nucleic acid (DNA).The results of these analyses have been calculated to nucleic acid content in μμg per microsporocyte, microspore or pollen grain, and the results related to logarithm of flower bud length, an index of the developmental status of the cells, and of their temporal relationship to meiosis, microspore mitosis and opening of the flower.DNA content per cell drops sharply at the end of meiosis, with the formation of four microspores from each microsporocyte. It then increases gradually during the microspore interphase between meiosis and the microspore mitosis. At microspore mitosis DNA content doubles rapidly. In the development of the resulting binucleate pollen grain, from microspore mitosis until the opening of the flower, there is a further gradual increase of DNA content. PNA content of these cells follows the same pattern up to microspore mitosis at a level about twice that of DNA, increases sharply at mitosis, and continues to increase rapidly at a rate nine times that for DNA in the maturing pollen grain.The absolute amounts of DNA and PNA are great. At the time of anthesis the two-celled pollen grain contains about 375 μμg of DNA and 1705 μμg of PNA.     

Volatile secretions and epicuticular hydrocarbons of the beetle Ulomoides dermestoides

Many species of tenebrionids produce and secrete a defensive volatile blend containing mainly benzoquinones and alkenes. In this study we characterized the volatile organic compounds (VOC) of the beetle Ulomoides dermestoides (Coleoptera: Tenebrionidae). Solid phase microextraction (SPME) coupled to capillary gas chromatography–mass spectrometry (CGC–MS) analysis was used to identify methyl-1,4-benzoquinone (MBQ), ethyl-1,4-benzoquinone (EBQ), 1-tridecene (C_13:1), and 1-pentadecene (C_15:1), representing more than 90% of the volatile blend. We also used CGC–MS to analyze the epicuticular hydrocarbons of U. dermestoides. Saturated, unsaturated, and branched structures with chain lengths ranging from 13 to 43 carbons were detected. n-pentacosane (C_25:0) and 9,11-pentacosadiene (9,11-C_25:2) were the most abundant components, representing more than 40% of the cuticular hydrocarbons.     

Maintenance of gametophytic development after symmetrical division in tobacco microspore culture

Isolated tobacco (Nicotiana tabacum L.) microspores maturing in vitro can be induced to undergo symmetrical divisions, instead of the normal asymmetrical first pollen mitosis, by addition of anther extracts to the culture medium. The two daughter cells in symmetrically divided pollen resemble vegetative pollen cells in cytological characteristics, nuclear size and chromatin condensation, are separated by a cell wall and remain viable during in vitro maturation. After transfer to a germination medium, only one of the two vegetativelike cells forms a pollen tube in vitro. Therefore, apparently normal gametophytic development can be maintained after symmetrical microspore division. These results are discussed in relation to current models for induction of microspore embryogenesis.     

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.     

Octad pollen formation in Cymbopetalum (Annonaceae): the binding mechanism

Our recent study of tetrad pollen formation in Annona (Annonaceae) revealed that after meiosis the callose-cellulose envelope forms a special conjugation with individual microspores and the forthcoming callose digestion is incomplete. The undigested part forms a central binder holding the four microspores of the tetrad together. This process causes the microspores to rotate 180 degrees. In this paper we describe pollen formation in another annonaceous genus, Cymbopetalum, in which the pollen is shed in octads, through use of light microscopy, epifluorescence microscopy, and TEM. In Cymbopetalum, two meiocytes, connected by abundant cytomictic channels, are produced in each sporangium. Octad pollen formation in Cymbopetalum is shown to be comparable to the synchronized formation of two connected Annona tetrads, which then integrate into a single octad. Unique features of Annona polyad formation, e.g. special binding between the callose-cellulose envelopes and microspores, incomplete callose digestion, and microspore rotation, also occur in Cymbopetalum. In addition, formation of the Cymbopetalum octad involves development of a cushion-like structure that binds the distal pronexine of all eight microspores, and there is the production of intine protrusions. The evolutionary origin of the callose-cellulose binding mechanism within the family is discussed.     

Arabidopsis mutant of AtABCG26, an ABC transporter gene, is defective in pollen maturation

In plants, pollen is the male gametophyte that is generated from microspores, which are haploid cells produced after meiosis of diploid pollen mother cells in floral anthers. In normal maturation, microspores interact with the tapetum, which consists of one layer of metabolically active cells enclosing the locule in anthers. The tapetum plays several important roles in the maturation of microspores. ATP-binding cassette (ABC) transporters are a highly conserved protein super-family that uses the energy released in ATP hydrolysis to transport substrates. The ABC transporter gene family is more diverse in plants than in animals. Previously, we reported that an Arabidopsis half-size type ABC transporter gene, COF1/AtWBC11/AtABCG11, is involved in lipid transport for the construction of cuticle layers and pollen coats in normal organ formation, as compared to CER5/AtWBC12/AtABCG12. However, physiological functions of most other ABCG members are unknown. Here, we identified another family gene, AtABCG26, which is required for pollen development in Arabidopsis. An AtABCG26 mutant developed very few pollen grains, resulting in a male-sterile phenotype. By investigating microspore and pollen development in this mutant, we observed that there was a slight abnormality in tetrad morphology prior to the formation of haploid microspores. At a later stage, we could not detect exine deposition on the microspore surface. During pollen maturation, many grains in the mutant anthers got aborted, and surviving grains were found to be defective in mitosis. Transmission of the mutant allele through male gametophytes appeared to be normal in genetic transmission analysis, supporting the view that the pollen function was disturbed by sporophytic defects in the AtABCG26 mutant. AtABCG26 can be expected to be involved in the transport of substrates such as sporopollenin monomers from tapetum to microspores, which both are plant-specific structures critical to pollen development.     

Cuticular hydrocarbons corroborate the distinction between lowland and highland Natal fruit fly (Tephritidae,Ceratitis rosa) populations

The cuticular hydrocarbons (CHs) and morphology of two Ceratitis rosa Karsch (Diptera: Tephritidae) populations, putatively belonging to two cryptic taxa, were analysed. The chemical profiles were characterised by two-dimensional gas chromatography with mass spectrometric detection. CHs of Ceratitis rosa that originated from the lowlands and highlands of Kenya comprised of n-alkanes, monomethylalkanes, dimethylalkanes and unsaturated hydrocarbons in the range of the carbon backbone from C₁₄ to C₃₇. Hydrocarbons containing C₂₉, C₃₁, C₃₃ and C₃₅ carbon atoms predominated in these two populations. 2-Methyltriacontane was the predominant compound in both populations. Quantitative differences in the distribution of hydrocarbons of different chain lengths, mainly the C₂₂, C₃₂, C₃₃ and C₃₄ compounds of these two populations, were observed despite indistinct qualitative differences in these hydrocarbons. Morphological analyses of male legs confirmed that the flies belong to different morphotypes of Ceratitis rosa previously labelled as R1 and R2 for lowland and highland populations, respectively. A statistical analysis of the CH compositions of the putative R1 and R2 species showed distinct interspecific identities, with several CHs specific for each of the lowland and highland populations. This study supports a hypothesis that the taxon Ceratitis rosa consists of at least two biological species.     

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。     

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.     

The occurrence of monounsaturated n-C21 and polyunsaturated C25 sedimentary hydrocarbons in the lipids of Antarctic marine organisms

Antarctic zooplankton have been found to be a potential source of sedimentary hydrocarbons. Monounsaturated C₂₁ n-alkenes and highly branched polyunsaturated C₂₅ n-alkenes were analysed in the aliphatic fraction of the lipids of Antarctic pelagic and inshore marine organisms. Cluster analysis of the species-based data set produced four main groups: phytoplankton, epipelagic herbivores, epipelagic carnivores and mesopelagic omnivores. The detailed pattern of alkenes exhibited differences within the groups and also with tissue type (krill, Euphausia superba). The origin of alkenes in Antarctic biota appeared to be either synthesis de novo or due to the condensation of smaller molecules. Formation of alkenes by the decarboxylation of fatty acids was not consistent with the hydrocarbon and fatty acid composition of Antarctic zooplankton. There was no evidence for direct assimilation of C₂₁ and C₂₅ alkenes by zooplankton or higher predators from their diet. Zooplankton C₂₅ alkenes are probably transported unaltered directly to the sediment as detritus or via predators in faecal material. Sedimentary C₂₅ alkenes are proposed as biomarkers of recent zooplankton activity in the water column.     

Hydroxylation of n-alkanes to secondary alcohols and their esterification in the grasshopper Melanoplus sanguinipes

Labeled n-alkanes administered to the grasshopper $\text{Melanoplus}$$\text{sanguinipes}$ are hydroxylated at or near the middle of the carbon chain. The secondary alcohols formed are then esterified. Chain length specificity is evident in both the hydroxylation of n-alkanes and the esterification of secondary alcohols, with the shorter chain C₂₃, C₂₁, C₁₉, and C₂₅ compounds converted to secondary alcohol wax esters more readily than the longer chain C₂₇, C₂₉, and C₃₁ compounds. Secondary alcohols and ketones are not reduced to alkanes.     

Pectin secretion and distribution in the anther during pollen development in Lilium

Using the monoclonal antibodies JIM 5 and 7, pectin was immunolocalized and quantitatively assayed in three anther compartments of Lilium hybrida during pollen development. Pectin levels in both the anther wall and the loculus increased following meiosis, were maximal during the early microspore stages and declined during the remainder of pollen ontogenesis. In the microspores/pollen grains, pectin was detectable at low levels during the microspore stages but accumulated significantly during pollen maturation. During early microspore vacuolation, esterified pectin epitopes were detected both in the tapetum cytoplasm and vacuoles. In the anther loculus, the same epitopes were located simultaneously in undulations of the plasma membrane and in the locular fluid. At the end of microspore vacuolation, esterified pectin epitopes were present within the lipids of the pollenkitt, and released in the loculus at pollen mitosis. Unesterified pectin epitopes were hardly detectable in the cytoplasm of the young microspore but were as abundant in the primexine matrix as in the loculus. During pollen maturation, both unesterified and esterified pectin labelling accumulated in the cytoplasm of the vegetative cell, concurrently with starch degradation. In the mature pollen grain, unesterified pectin epitopes were located in the proximal intine whereas esterified pectin epitopes were deposited in the distal intine. These data suggest that during early microspore development, the tapetum secretes pectin, which is transferred to the primexine matrix via the locular fluid. Further, pectin is demonstrated to constitute a significant component of the pollen carbohydrate reserves in the mature grain of Lilium. Received: 3 July 2000 / Accepted: 19 October 2000     

Effects of aluminum on DNA synthesis, cellular polyamines, polyamine biosynthetic enzymes and inorganic ions in cell suspension cultures of a woody plant, Catharanthus roseus

Individual buds of Brassica napus cv. Topas, near the first pollen mitosis, were used for microspore culture. Bud and petal lengths were recorded. Microspores isolated from the individual buds were plated and small samples were fixed for cytology. Following embryo induction and three weeks of culturing, numbers of embryos were scored. Bud and petal lengths did not accurately indicate which buds would supply microspores that would form embryos at high frequencies. Fluorescence microscopy was used to examine nuclei stained with Hoechst 33258 and vacuolar morphology of microspores was revealed by the weaker fluorescence due to glutaraldehyde fixation. Following isolation, nuclear and vacuolar characteristics were used to stage the microspores as miduninucleate, late uninucleate vacuolate, late uninucleate, mitotic, or binucleate. The relationship of developmental stage to the frequency of microspore-derived embryos was evaluated. A classification scheme was developed which uses the relative proportions of microspores at each of the stages to identify microspore isolations that would form embryos at high frequencies. It was found that when 1 to 87% of the isolated microspores were binucleate, 21.4 ± 3.0% of the viable microspores developed into embryos. This was a significant ( P < 0.001) increase over the other 3 classes. The ability to select highly embryogenic microspore isolations is of great advantage for developmental cell biology studies.     

A glycine-rich protein that facilitates exine formation during tomato pollen development

Formation of the unique and highly diverse outer cell wall, or exine, of pollen is essential for normal pollen function and survival. However, little is known about the many contributing proteins and processes involved in the formation of this wall. The tomato gene LeGRP92 encodes for a glycine-rich protein produced specifically in the tapetum. LeGRP92 is found as four major forms that accumulate differentially in protein extracts from stamens at different developmental stages. The three largest molecular weight forms accumulated during early microspore development, while the smallest molecular weight form of LeGRP92 was present in protein extracts from stamens from early microsporogenesis through anther dehiscence, and was the only form present in dehisced pollen. Light microscopy immunolocalization experiments detected LeGRP92 at only two stages, late tetrad and early free microspore. However, we observed accumulation of the LeGRP92 at the early tetrad stage of development by removing the callose wall from tetrads, which allowed LeGRP92 detection. Transmission electron microscopy confirmed the LeGRP92 accumulation from microspore mother cells, tetrads through anther dehiscence. It was observed in the callose surrounding the microspore mother cells and tetrads, the exine of microspores and mature pollen, and orbicules. Plants expressing antisense RNA had reduced levels of LeGRP92 mRNA and protein, which correlated to pollen with altered exine formation and reduced pollen viability and germination. These data suggest that the LeGRP92 has a role in facilitating sporopollenin deposition and uniform exine formation and pollen viability.     

Pollen vacuoles and their significance

Vacuoles of several types can be observed in pollen throughout its development. Their physiological significance reflects the complexity of the biological process leading to functional pollen grains. Vacuolisation always occurs during pollen development but when ripe pollen is shed the extensive translucent vacuoles present in the vegetative parts in previous stages are absent. Vacuole functions vary according to developmental stage but in ripe pollen they are mainly storage sites for reserves. Vacuoles cause pollen to increase in size by water accumulation and therefore confer some degree of resistance to water stress. Modalities of vacuolisation occur in pollen in the same manner as in other tissues. In most cases, autophagic vacuoles degrade organelles, as in the microspore after meiosis, and can be regarded as cytoplasm clean-up following the transition from the diploid sporophytic to the haploid gametophytic state. This also occurs in the generative cell but not in sperm cells. Finally, vacuoles have a function when microspores are used for pollen embryogenesis in biotechnology being targets for stress induction and afterwards contributing to cytoplasmic rearrangement in competent microspores.