Anther starch variations in Lilium during pollen development

Starch was cytologically localized and biochemically assayed in different anther cell layers of Lilium cv. Enchantment during pollen development and its presence was correlated with anther growth. Two phases could be distinguished: the first, the growth phase, extends from the beginning of meiosis to the vacuolated microspore stage and corresponds to maximum increase in anther size and weight. During this period, microspores lack amyloplasts and starch is degraded in the outer staminal wall layers. The tapetum does not contain starch reserves but accumulates a PAS-positive substance in its vacuole. The second phase, the maturation phase, begins with the late vacuolated microspore stage and lasts until pollen maturation. Anther growth is slowed during this phase. A wave of amylogenesis/ amylolysis occurs first in the late vacuolated-microspores and young pollen grains and, next, in the staminal envelopes. In the pollen grain, the cytoplasm of the vegetative cell is filled with starch, but amyloplasts are not detected in the generative cell. When pollen grains ripen, amylaceous reserves are replaced with lipids. In the staminal envelopes, the second amylogenesis is particularly evident in the endothecium and the middle layers; the peak of starch is reached at the young bicellular pollen grain stage; starch disappears from the anther wall early during the maturation phase. The wave of amylogenesis/amylolysis occurring in the staminal envelopes during the maturation phase is peculiar to Lilium. It is interpreted as a sudden increase in carbohydrate level caused by lower anther needs when the growth is completed. Staminal envelopes may act as a physiological buffer and regulate soluble sugar level in the anther. Stages of anther growth correlate with starch content variations and this suggests that during the growth phase, products of starch hydrolysis in the staminal envelopes may be consumed partly by anther cell layers and partly by microspores.     

玉竹雄配子的发育——着重阐明质体在生殖细胞和营养细胞中的分布和变化

玉竹(Polygonatum simizui Kitag)小孢子在分裂前,质体极性分布导致分裂后形成的生殖细胞不含质体,而营养细胞包含了小孢子中全部的质体。生殖细胞发育至成熟花粉时期,及在花粉管中分裂形成的两个精细胞中始终不含质体。虽然生殖细胞和精细胞中都存在线粒体,但细胞质中无DNA类核。玉竹雄性质体的遗传为单亲母本型。在雄配子体发育过程中,营养细胞中的质体发生明显的变化。在早期的营养细胞质中,造粉质体增殖和活跃地合成淀粉。后期,脂体增加而造粉质体消失。接近成熟时花粉富含油滴。对百合科的不同属植物质体被排除的机理及花粉中贮藏的淀粉与脂体的转变进行了讨论。     

Plastid differentiation during androgenesis in albino and non-albino producing cultivars of barley (Hordeum vulgare L.)

In order to better understand androgenic albinism in barley, we compared plastid differentiation during anther culture in two cultivars, an albino (spring cultivar Cork) and a non-albino (winter cultivar Igri) producing cultivar. The ultrastructure of plastids and the relative amount of DNA containing plastids were followed in both cultivars during the androgenic process and correlated with the proportion of regenerated chlorophyllous plantlets. For androgenesis, anthers were collected at the uninucleate stage, during mid- or late-microspore vacuolation. At this stage DNA was detected in 15.3 ± 2. 7% of microspore plastid sections in the winter cultivar Igri, compared to 1.7 ± 0.5% in the spring cultivar Cork. In the winter cultivar Igri, starch was broken down after anther pretreatment but plastids divided rapidly during anther culture and thylakoids developed in the stroma. Prior to regeneration, plastids contained 2.0 ± 0.2 thylakoids per plastid and starch represented 26.1 ± 3.3% of the plastid volume. In the spring cultivar Cork, plastids followed a different developmental pathway. After anther pretreatment, microspore plastids differentiated exclusively into amyloplasts, accumulating starch and losing their thylakoids as well as their capacity to divide. This developmental pattern became progressively more marked, so that by the end of anther culture plastids contained 0.5 ± 0.4 thylakoids per plastid and starch represented up to 90.3 ± 4.3% of plastid volume. Following androgenesis, the response was similar in both cultivars except that the winter cultivar Igri provided 87.8% of chlorophyllous plantlets compared to 99.7% albino plantlets in the cultivar Cork. The results presented here suggest that the exclusive regeneration of albino plantlets in the spring cultivar Cork may be due to degradation of microspore plastid DNA during early pollen development, preventing the plastids from differentiating into chloroplasts under culture conditions. Received: 13 March 2000 / Revision accepted: 6 June 2000     

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.     

Development of Plastids in Pollen and Tapetum of Rye-grass, Lolium perenne L

Proplastids of both tapetal cells and microsporocytes were presentearly in anther development. Tapetal proplastids differentiated—probablyinto elaioplasts—at late microspore stage. The tapetalcytoplasm was completely resorbed by early tricellular pollenstage. Microspore proplastids differentiated into amyloplastsat early bicellular stage, and were present in both vegetativeand generative cells. In the generative cell, the amyloplastswere ephemeral and apparently degenerated within autophagicvacuoles. Plastids were absent from sperm cells. Vegetativecell amyloplasts increased in number apparently by fission suchthat one amyloplast produced one amyloplast and one proplastidper division. Mature pollen grains were estimated to containbetween 550 and 820 amyloplasts with only one starch granuleper plastid. Elaioplasts, amyloplasts, plastid division, plastid differentiation, starch granules, autophagy, Lolium perenne, Poaceae, rye-grass     

红盖鳞毛蕨孢子囊早期发育与质体分化的研究

利用光学显微镜和透射电子显微镜观察了红盖鳞毛蕨(Dryopteris erythrosora(Eaton)O.Ktze.)孢子囊的发育及在此期间质体的分化过程。研究表明:(1)红盖鳞毛蕨孢子囊的发育类型属于薄囊蕨型;(2)绒毡层为混合型,即内层绒毡层为原生质团型,外层绒毡层为腺质型;(3)孢子囊原始细胞中的质体通过3条路径分化,其一,原始细胞中含淀粉粒的质体通过分裂分配到下方细胞,继而进入孢子囊柄;其二,原始细胞分裂产生的新生质体被分配到上方细胞,进而被分配到除顶细胞外的原基细胞中,顶细胞将含淀粉粒的质体通过分裂分配到外套层原始细胞中;其三,顶细胞也将具淀粉粒的质体通过分裂分配到内部细胞,使分裂产生的孢原细胞和绒毡层原始细胞具新生质体;造孢细胞和孢子母细胞的质体具淀粉粒,孢子母细胞还具油体,新生孢子中具造粉体和油体;两层绒毡层具新生质体,随着退化外层绒毡层出现造粉体,内层绒毡层出现油体;(4)红盖鳞毛蕨与少数被子植物小孢子发育阶段质体分化模式类似,由前质体分化为造粉体再到油体。研究结果为蕨类植物质体在孢子囊发育过程不同组织细胞中的差异分化提供了新观察资料,为蕨类植物发育生物学和系统演化研究提供科学依据。     

Control of the developmental pathway of tobacco pollen in vitro

We developed a new method for culture of isolated pollen. Using highly homogeneous populations of immature pollen grains of Nicotiana tabacum L. prepared by means of Percoll density gradient centrifugation, we could direct their developmental pathway by regulating certain culture conditions. When the pollen population was cultured in basal medium with glutamine, most pollen grains underwent normal maturation. On the other hand, when first cultured in basal medium without glutamine, most pollen grains did not mature but after transfer to medium with glutamine and sucrose began to divide. This method for inducing pollen cell division was possible only with midbinucleate pollen grains which are characterized by having no central vacuole and no or only a few starch grains. Evidently, some essential changes necessary for the embryogenic response can be induced by glutamine starvation only in pollen grains at a specific stage.     

Microspore development inBrassica napus and the effect of high temperature on division in vivo and in vitro

Summary Brassica napus cv. Topas microspores isolated and cultured near the first pollen mitosis and subjected to a heat treatment develop into haploid embryos at a frequency of about 20%. In order to obtain a greater understanding of the induction process and embryogenesis, transmission electron microscopy was used to study the development of pollen from the mid-uninucleate to the bicellular microspore stage. The effect of 24 h of high temperature (32.5 °C) on microspore development was examined by heat treating microspore cultures or entire plants. Mid-uninucleate microspores contained small vacuoles. Late-uninucleate vacuolate microspores contained a large vacuole. The large vacuole of the vacuolate stage was fragmented into numerous small vacuoles in the late-uninucleate stage. The late-uninucleate stage contained an increased number of ribosomes, a pollen coat covering the exine and a laterally positioned nucleus. Prior to the first pollen mitosis the nucleus of the lateuninucleate microspore appeared to be appressed to the plasma membrane; numerous perinuclear microtubules were observed. Microspores developing into pollen divided asymmetrically to form a large vegetative cell with amyloplasts and a small generative cell without plastids. The cells were separated by a lens-shaped cell wall which later diminished. At the late-bicellular stage the generative cell was observed within the vegetative cell. Starch and lipid reserves were present in the vegetative cell and the rough endoplasmic reticulum and Golgi were abundant. The microspore isolation procedure removed the pollen coat, but did not redistribute or alter the morphology of the organelles. Microspores cultured at 25 °C for 24 h resembled late-bicellular microspores except more starch and a thicker intine were present. A more equal division of microspores occurred during the 24 h heat treatment (32.5 °C) of the entire plant or of cultures. A planar wall separated the cells of the bicellular microspores. Both daughter cells contained plastids and the nuclei were of similar size. Cultured embryogenie microspores contained electron-dense deposits at the plasma membrane/cell wall interface, vesicle-like structures in the cell walls and organelle-free regions in the cytoplasm. The results are related to embryogenesis and a possible mechanism of induction is discussed.Abbreviations B binucleate - LU late uninucleate - LUV late uninucleate vacuolate - M mitotic - MU mid-uninucleate - RER rough endoplasmic reticulum - TEM transmission electron micrograph     

雄性不育无籽刺梨花药发育过程中生理特性的初步研究

为探究无籽刺梨(Rosasterilis)雄性不育原因,采用1%I2-KI染色法观察花粉活性,并对无籽刺梨和正常可育刺梨(R.roburghii)花药不同发育时期的生理生化指标进行了研究。结果表明,无籽刺梨的败育花粉占95.5%,刺梨的正常花粉占99%。刺梨花药的可溶性蛋白质、可溶性糖和脯氨酸含量在各时期的总体变化趋势相似,可溶性淀粉含量呈上升趋势,而无籽刺梨花药的可溶性蛋白质、可溶性糖、淀粉和脯氨酸含量在各时期的变化无规律可循,且花粉成熟期这4种物质的含量均明显低于刺梨,即花粉成熟期缺少各营养物质的积累。在花药发育过程中,无籽刺梨的SOD活性均低于刺梨;MDA含量呈上升趋势,且上升幅度比刺梨大;MDA含量和POD活性均高于刺梨。因此,营养物质的匮乏和酶系统的紊乱可能是导致无籽刺梨雄性不育的原因。     

Ultrastructural features of the starch sheath cells of the primary pulvinus after gravistimulation of the sensitive plant (Mimosa pudica L.)

Summary InMimosa pudica the primary and secondary motor organs (pulvini) of fully grown leaves are capable of graviresponse. These organs possess sedimentable amyloplasts in their starch sheath cells.In the primary pulvinus these cells are characterized by a structural polarity induced by the localization of nucleus at their (morphologically) apical part and the localization of amyloplasts at their (physically) basal part. These cells also display structural peculiarities including plasmodesmatal disposition, little development of the endoplasmic reticulum and an absence of vacuolar tannins; moreover, the sedimentation of the amyloplasts, induced by gravistimulation, is accompanied by the variation of localization of the cytoplasm, vacuole and mitochondria and by structural modifications of the nucleus and endoplasmic reticulum.     

Cytological evidence of biparental inheritance of plastids and mitochondria inPelargonium

Summary Based on the organelle differences between egg and sperm cells inPelargonium hortorum, the zygote, proembryo, and endosperm were examined under the transmission electron microscope. Plastids and mitochondria in the egg cell are significantly different from those of the sperm cell. Egg plastids are starch-containing and less electron dense. They appear circular, elliptical irregular elongate in sections. Sperm cell plastids are relatively electrondense, mostly cup-shaped or dumbbell and devoid of starch granules. Mitochondria of the egg cell are giant and mostly cup-shaped while sperm mitochondria are smaller and usually circular in section. Double fertilization is completed by 24 h after pollination and the pollen tube can be seen in the degenerated synergid. In the zygote, plastids and mitochondria from male and female gametes can be distinguished by their characteristic differences. Moreover, paternal and maternal organelles appear to be distributed at random in the zygote. Aside from the pollen tube and its released starch granules, there is no enucleated cytoplasmic body in the degenerated synergid. Two days after pollination, the zygote undergoes one transverse division to form a 2-celled proembryo which consists of one larger vacuolated basal cell and one smaller densely cytoplasmic apical cell. Paternal and maternal organelles can be detected in both cells of the proembryo and also in the endosperm at this stage. From these results, it can be concluded that plastids and mitochondria from both male and female gametes have been transmitted into the apical cell of the proembryo and most probably to the following generation.Abbreviations TEM transmission electron microscope - DAPI 4,6-diamidino-2-phenylindole - RFLP restriction fragment length polymorphism     

Structural changes in the plastid DNA of rice (Oryza sativa L.) during tissue culture

To investigate the rearrangement of the plastid genome during tissue culture, DNA from rice callus lines, which had been derived individually from single protoplasts isolated from seed or pollen callus (protoclones), was analyzed by Southern hybridization with rice chloroplast DNA (ctDNA) clones as probes. Among 44 long-term cultured protoclones, maintained for 4, 8 or 11 years, 28 contained plastid DNA (ptDNA) from which portions had been deleted. The ptDNA of all protoclones that had been maintained for 11 years had a deletion that covered a large region of the plastid genome. The deletions could be classified into 15 types from their respective sizes and positions. By contrast, no deletions were found in the ptDNA of 38 protoclones that had been maintained for only 1 month. These results indicate that long-term culture causes deletions in the plastid genome. Detailed hybridization experiments revealed that plastid genomes with deletions in several protoclones were organized as head-to-head or tail-to-tail structures. Furthermore, ptDNAs retained during long-term culture all had a common terminus at one end, where extensive rearrangement is known to have occurred during the speciation of rice and tobacco. Morphological analysis revealed the accumulation of starch granules in plastids and amyloplasts in protoclones in which the plastid genome had undergone deletion. Our observations indicated that novel structural changes in the plastid genome and morphological changes in the plastid had occurred in rice cells during long-term tissue culture. Moreover, the morphological changes in plastids were associated with deletions in the plastid genome.     

Pollen tube reuses intracellular components of nucellar cells undergoing programmed cell death in Pinus densiflora

Through the process known as programmed cell death (PCD), nucelli of Pinus densiflora serve as the transmitting tissue for growth of the pollen tube. We sought to clarify the processes of degradation of nucellar cell components and their transport to the pollen tube during PCD in response to pollen tube penetration of such nucelli. Stimulated by pollination, synthesis of large amounts of starch grains occurred in cells in a wide region of the nucellus, but as the pollen tube penetrated the nucellus, starch grains were degraded in amyloplasts of nucellar cells. In cells undergoing PCD, electron-dense vacuoles with high membrane contrast appeared, assumed a variety of autophagic structures, expanded, and ultimately collapsed and disappeared. Vesicles and electron-dense amorphous materials were released inside the thickened walls of cells undergoing PCD, and those vesicles and materials reaching the pollen tube after passing through the extracellular matrix were taken into the tube by endocytosis. These results show that in PCD of nucellar cells, intracellular materials are degraded in amyloplasts and vacuoles, and some of the degraded material is supplied to the pollen tube by vesicular transport to support tube growth.     

Factors affecting in vitro maturation of isolated maize microspores

Summary An in vitro method to simulate pollen development was developed in maize (Zea mays L.). Microspores at the late uninucleate to early binucleate stage were isolated and cultured under various conditions. Cell viability, starch content and the formation of the three nuclei as found in normal mature pollen were monitored during the course of the culture. Media composition was modified in order to promote starch accumulation and frequency of mitosis, while maintaining the viability of the microspores. Under the best conditions, up to 12% of the microspores matured in vitro into trinucleate, starch-filled viable pollen grains which were unable to germinate or produce seeds. At different stages during in vitro maturation, proteins patterns were analyzed and compared with their in vivo equivalent and the patterns were only partially similar.     

Dynamics of vegetative cytoplasm during generative cell formation and pollen maturation inArabidopsis thaliana

Summary Ultrastructural changes of pollen cytoplasm during generative cell formation and pollen maturation inArabidopsis thaliana were studied. The pollen cytoplasm develops a complicated ultra-structure and changes dramatically during these stages. Lipid droplets increase after generative cell formation and their organization and distribution change with the developmental stage. Starch grains in amyloplasts increase in number and size during generative and sperm cell formation and decrease at pollen maturity. The shape and membrane system of mitochondria change only slightly. Dictyo-somes become very prominent, and numerous associated vesicles are observed during and after sperm cell formation. Endoplasmic reticulum appears extensively as stacks during sperm cell formation. Free and polyribosomes are abundant in the cytoplasm at all developmental stages although they appear denser at certain stages and in some areas. In mature pollen, all organelles are randomly distributed throughout the vegetative cytoplasm and numerous small particles appear. Organization and distribution of storage substances and appearance of these small particles during generative and sperm cell formation and pollen maturation are discussed.     

Anther culture of Sorghum bicolor (L.) Moench

The sequence of pollen development from the tetrad stage to the mature tricellular grain was studied in freshly harvested anthers of Sorghum bicolor. This pattern of development was then compared with that occurring during panicle pretreatment and subsequent anther incubation in vitro. It was found that during pretreatment at 7° C mitoses of the vegetative cell were induced in up to 30% of the pollen. During anther incubation procallus development was highly polarised with contributions from both the generative and vegetative cells. After pretreatment at 14 or 20° C the generative cell became detached from the pollen wall and it was not possible to determine whether subsequent development involved only the vegetative cell or both the vegetative and generative cells.Although retarded pollen grains were observed both in vivo and in vitro, and were occasionally seen to divide in culture, they did not appear to be the source of the procalluses produced.     

Detection of brassinosteroids in pollen of Lolium perenne L. by immunocytochemistry

Bioactive brassinosteroids have been localized in developing and mature pollen of anhydrously fixed rye-grass (Lolium perenne) by immunocytochemistry using polyclonal antibodies to castasterone generated in rabbits. Tricellular pollen fixed by freeze-substitution was also labelled in the starch granules. Study of the developmental sequence of the pollen through the microsporocyte, microspore, bicellular and tricellular stages showed that the brassinosteroids were increasingly sequestered in starch granules as the amyloplasts matured, supporting the view that these are storage organelles for these potent plant growth promoters. In bicellular pollen, heavy labelling was seen in the zone within 0.5 m of the starch granule, where stromal tissue remains. Thus, the stroma may be the site of synthesis of these compounds. During aqueous fixation, the brassinosteroids leached from the starch granules of tricellular pollen, indicating that they would be quickly available after imbibition to influence the physiology of germinating pollen. The results from high-performance liquid chromatography of dansylaminophenylboronates from partially purified extracts of freshly dehisced tricellular pollen of rye-grass showed 25-methylcastasterone may be a minor component, together with two unknown peaks. No specific binding of brassinolide to any soluble proteins extracted from tricellular rye-grass pollen was observed using the antibodies in gel electrophoresis or enzyme-linked immunosorbent assays.Abbreviations HPLC high-performance liquid chromatography - R_t retention time - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We thank the Australian Research Council for support, the N.S.W. Department of Agriculture for rice seed, Professor K. Mori and the Zen-Noh Corporation for authentic brassinosteroid samples, Dr. I. Hudson for statistical advice, Dr. A. Bacic and Ms. I. Bonig for helpful discussion, and J.M.S. thanks Professor R.B. Knox for laboratory facilities.     

Drought-induced male sterility in rice: Changes in carbohydrate levels and enzyme activities associated with the inhibition of starch accumulation in pollen

Male reproductive development of rice (Oryza sativa L.) is very sensitive to drought. A brief, transitory episode of water stress during meiosis in pollen mother cells of rice grown under controlled environmental conditions induced pollen sterility. Anthers containing sterile pollen were smaller, thinner, and often deformed compared to normal anthers of well-watered plants. Only about 20% of the fully developed florets in stressed plants produced grains, compared to 90% in well-watered controls. Water stress treatments after meiosis were progressively less damaging. Levels of starch and sugars and activities of key enzymes involved in sucrose cleavage and starch synthesis were analyzed in anthers collected at various developmental stages from plants briefly stressed during meiosis and then re-watered. Normal starch accumulation during pollen development was strongly inhibited in stress-affected anthers. During the period of stress, both reducing and non-reducing sugars accumulated in anthers. After the relief of stress, reducing sugar levels fell somewhat below those in controls, but levels of non-reducing sugars remained higher than in controls. Activities of acid invertase and soluble starch synthase in stressed anthers were lower than in controls at comparable stages throughout development, during as well as after stress. Stress had no immediate effect on ADP-glucose pyrophosphorylase activity, but had an inhibitory aftereffect throughout post-stress development. Sucrose synthase activity, which was, relatively speaking, much lower than acid invertase activity, was only slightly suppressed by stress. The results show that it is unlikely that pollen sterility, or the attendant inhibition of starch accumulation, in water-stressed rice plants are caused by carbohydrate starvation per se. Instead, an impairment of enzymes of sugar metabolism and starch synthesis may be among the potential causes of this failure.     

Occurrence of a Putative Precursor to the Amyloplast,the “Amyloplast Initial” in Developing Stolons of the Potato

Cells at the apical part of developing stolons of the potato (Solatium tuberosum L. cv. Norin 1) were analyzed for the occurrence of putative precursors to amyloplasts, designated “amyloplast initial.” Ultrastructural studies showed that the cells contained the expected novel organelle. It was about 1 μm in diameter, devoid of thylakoid membranes, and was stained to a similar extent as the stroma of amyloplasts by uranyl acetate and lead citrate. Formation of thylakoid membranes and starch granules takes place at an early stage of development of these initials when they are just a few μm in diameter. At this stage, proliferation of the initials takes places by division at random sites.