Secretory tapetum of Brassica oleracea L.: polarity and ultrastructural features

Summary The ultrastructure of the secretory, binucleate tapetum of Brassica oleracea in the micro spore mother cell (MMC) stage through to the mature pollen stage is reported. The tapetal cells differentiate as highly specialized cells whose development is involved in lipid accumulation in their final stage. They start breaking down just before anther dehiscence. Nuclei with dispersed chromatin, large nucleoli and many ribosomes in the cytoplasm characterize the tapetal cells. The wall-bearing tapetum phase ends at the tetrade stage. The dissolution of tapetal walls begins from the inner tangential wall oriented towards the loculus and proceeds gradually along the radial walls to the outer tangential one. The plasmodesmata transversing the radial walls between tapetal cells persist until the mature microspore, long after loss of the inner tangential wall. After wall dissolution, the tapetal protoplasts retain their integrity and position within the anther locule. The tapetal cell membrane is in direct contact with the exine of the microspores/pollen grains and forms tubular evaginations that increase its surface area and appear to be involved in the translocation of solutes from the tapetal cells to the microspores/ pollen grains. The tapetal cells exhibit a polarity expressed by spatial differentiation in the radial direction.     

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     

Development of embryos and seedlings from pollen grains inLycium halimifolium Mill. in thein vitro culture

Anthers ofLycium halimifolium were grownin vitro at the following stages of development: tetrads, microspores and binucleate pollen grains. Pollen plantlets were obtained only from anthers inoculated at the stage of microspores. The growth of androgenic embryos was very slow. Mature embryos were formed in about 3% of the inoculated anthers. The largest number of embryos obtained from one anther was 4.     

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.     

Reproduction in seagrasses: pollen wall morphogenesis in Amphibolis antarctica and wall structure in filiform grains

The pre–meiotic anther of the marine angiosperm Amphibolis antarctica contains microsporocytes and sterile cells. The microsporocytes divide conventionally to produce tetrads, but the sterile cells degenerate and contribute to the future pe–riplasmodium. Each tetrad of young microspores is contained within a vesicle defined by a membrane. After release from the tetrad, the microspores increase in length and rapidly become filiform. The microspore nucleus soon divides and partitioning of the cytoplasm delimits the generative cell from the vegetative cell of the binucleate pollen grain. The division and the early pollen growth occurs while the grains are segregated within vesicles in the periplasmodium. These compartments, established at microspore release, remain structurally intact throughout the vacuolate period of pollen development, when pollen wall assembly begins. This process is initiated as particles migrate from the inner face of the vesicle membrane into the lumen of the vesicle and microfibrillar elements form between adjacent particles. The particles and microfibrils form a loose, three–dimensional network. The vesicle membrane then disappears and the binuclate grains become immersed in the tapetal residuum. Additional wall components are now deposited upon the primary fibrillar stratum. Short lamellae, resembling fragments of membrane, frequently associated with electron–opaque globuli, are found intermixed with the surface microfibrils. Apparently, granular material originating in the degenerating periplasmodium may be the precursor of the globuli, and contact with the lamellae brings about an alteration in state. At this stage the pollen wall is resolved as two distinct fibrillar strata and the lamellae and globuli are incorporated as inclusions into the superficial zone of the outer stratum. The mature pollen wall exhibits faint stratification and the presence of the subsurface inclusions is readily demonstrated in germinating grains by section staining with phosphotungstic acid. The pollen wall in A. antarctica is compared with that in filiform grains of other seagrasses.     

FINE STRUCTURAL STUDIES OF ZEA MAYS POLLEN I: CELL MEMBRANES AND EXINE ONTOGENY

Electron microscopy was used to study pollen wall ontogeny in Zea mays. The initial stage of development consisted of compartmentalization of microspores within callose special walls. Microspore plasma membranes retracted and tubular elements of the endoplasmic reticulum became perpendicularly oriented to the plasma membranes. Evaginations of the endoplasmic reticulum into the microspore plasma membrane resulted in the establishment of a template or blueprint of the mature pollen wall. Sporopollenin deposition upon the template began immediately after dissolution of the callose special walls and release of the microspores into the anther locule. The columellae were the first pollen wall units to be formed; the tectum and foot layer became established shortly thereafter. The granular endexine was the last-formed unit. The relationships of membrane systems to the ontogeny of the pollen wall units and the mode of pollen wall growth are discussed.     

Cold-induced repression of the rice anther-specific cell wall invertase gene OSINV4 is correlated with sucrose accumulation and pollen sterility

Low temperatures during rice (Oryza sativa L.) pollen development cause pollen sterility and decreased grain yield. We show that the time of highest sensitivity to cold coincides with the time of peak tapetal activity: the transition of the tetrad to early uni-nucleate stage (young microspore, YM stage). Low temperatures at this stage of pollen development result in an accumulation of sucrose in the anthers, accompanied by decreased activity of cell wall bound acid invertase and depletion of starch in mature pollen grains. Expression analysis of two cell wall (OSINV1, 4) and one vacuolar (OSINV2) acid invertase genes showed that OSINV4 is anther-specific and down-regulated by cold treatment. OSINV4 is transiently expressed in the tapetum cell layer at the YM stage, and later from the early binucleate stage in the maturing microspores. The down-regulation of OSINV4 expression in the tapetum at YM may cause a disruption in hexose production and starch formation in the pollen grains. In a cold-tolerant cultivar, OSINV4 expression was not reduced by cold; sucrose did not accumulate in the anthers and starch formation in the pollen grains was not affected.     

Development and cytochemistry of pollen and tapetum in Mitriostigma axillare (Rubiaceae)

The development of pollen grains and tapetum in Mitriostigma axillare (Rubiaceae) was studied from anther primordium to dehiscence. Anthers were freeze-cracked and studied with SEM. Embedded anthers were sectioned and studied with LM and TEM. Cytochemistry was performed in order to distinguish the different layers of the sporoderm and to determine its chemical nature at different development stages. The pollen grains remained as tetrads by partial fusion of the exine, probably because of reduced callose septa during the stage of microspore tetrads within callose envelopes. Characteristic features of the sporoderm were an irregular foot layer, an endexine composed of amalgamated granules, a transient granular-fibrous layer beneath the endexine, and a thin intine. During maturation of the exine, the endexine became chemically different from the ectexine. All layers of the sporoderm were reduced in thickness due to stretching during the engorgement of the pollen grains prior to dehiscence. The pollen grains were colpoidorate with a reticulate to microreticulate tectum covered with a scanty surface coating. The mature pollen grains were binucleate and contained a lot of starch grains. Thick intineous onci protruded through the apertures and formed papillae. About 50% of the microspores were aborted. The tapetum was of secretory type, probably with cycles of hyperactivity and protrusions of the cells into the locular cavity. No syncytium was formed and there were neither orbicules nor tapetal membrane.     

Microscopy of a cytoplasmic male-sterile soybean from an interspecific cross between Glycine max and G. soja (Leguminosae)

Cytoplasmic male sterility has been found independently in soybean three times since 1995, but no microscopic investigation has been published. The purpose of this microscopic study was to establish the developmental sequence leading to sterility in a cytoplasmic male-sterile soybean line that has been found to be stable under all environmental conditions tested and to demarcate the temporal and spatial parameters that result in degeneration of the microspores and pollen grains. Light microscopy showed an abnormal development and/or premature degeneration of the tapetum after meiosis II, but some pollen grains persisted until after microspore mitosis. The pollen grains never completely filled with reserves. Premature formation of the endothecium also was evident. Histochemical staining for water-insoluble carbohydrates revealed an abnormal pattern of starch deposition in anther walls that coincided with lack of pollen filling. Electron microscopy showed degeneration of the inner mitochondrial membrane in the tapetal cells as the first detectable change leading to cell degeneration. Subsequently, the tapetal endoplasmic reticulum exhibited atypical concentric rings. Pollen grains displayed mitochondria with unusually enlarged inner mitochondrial spaces, degraded plastids, a rudimentary intine, and no starch or lipid reserves. Results link mitochondrial degeneration, premature formation of the endothecium, and energy deprivation to male sterility.     

Development of the pollen grain and tapetum of wheat (Triticum aestivum) in untreated plants and plants treated with chemical hybridizing agent RH0007

Summary A study of pollen development in wheat was made using transmission electron microscopy (TEM). Microspores contain undifferentiated plastids and mitochondria that are dividing. Vacuolation occurs, probably due to the coalescence of small vacuoles budded off the endoplasmic reticulum (ER). As the pollen grain is formed and matures, the ER becomes distended with deposits of granular storage material. Mitochondria proliferate and become filled with cristae. Similarly, plastids divide and accumulate starch. The exine wall is deposited at a rapid rate throughout development, and the precursors appear to be synthesized in the tapetum. Tapetal cells become binucleate during the meiosis stage, and Ubisch bodies form on the plasma membrane surface that faces the locule. Tapetal plastids become surrounded by an electron-translucent halo. Rough ER is associated with the halo around the plastids and with the plasma membrane. We hypothesize that the sporopollenin precursors for both the Ubisch bodies and exine pollen wall are synthesized in the tapetal plastids and are transported to the tapetal cell surface via the ER. The microspore plastids appear to be involved in activities other than precursor synthesis: plastid proliferation in young microspores, and starch synthesis later in development. Plants treated with the chemical hybridizing agent RH0007 show a pattern of development similar to that shown by untreated control plants through the meiosis stage. In the young microspore stage the exine wall is deposited irregularly and is thinner than that of control plants. In many cases the microspores are seen to have wavy contours. With the onset of vacuolation, microspores become plasmolyzed and abort. The tapetal cells in RH0007-treated locules divide normally through the meiosis stage. Less sporopollenin is deposited in the Ubisch bodies, and the pattern is less regular than that of the control. In many cases, the tapetal cells expand into the locule. At the base of one of the locules treated with a dosage of RH0007 that causes 95% male sterility, several microspores survived and developed into pollen grains that were sterile. The conditions at the base of the locule may have reduced the osmotic stress on the microspores, allowing them to survive. Preliminary work showed that the extractable quantity of carotenoids in RHOOO7-treated anthers was slightly greater than in controls. We concluded that RH0007 appears to interfere with the polymerization of carotenoid precursors into the exine wall and Ubisch bodies, rather than interfering with the synthesis of the precursors.     

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。     

红麻细胞质雄性不育系与保持系花药活性氧代谢差异比较

以红麻细胞质雄性不育系L23A及其保持系L23B为材料,比较其花药淀粉及可溶性糖含量变化并分析呼吸速率、活性氧产生速率、丙二醛(MDA)含量以及活性氧清除酶(POD、SOD)含量变化,来探讨活性氧伤害与红麻雄性不育的关系。结果表明:在小孢子发育的单核期,不育系呼吸速率与保持系差异不明显,但不育系花药O^-₂·含量高于保持系; 在双核期,不育系的呼吸速率明显低于其保持系,但不育系花药O^-₂·含量与保持系花药相近; 不育系在单核期和双核期的呼吸速率几乎没有变化,而保持系同一时期的呼吸速率呈明显增高趋势; 在不育系败育过程中,药隔维管组织中的大颗粒淀粉含量几乎不变,且不育系花药中的可溶性糖含量在单核期和双核期均低于保持系。推测是由于不育系花药中抗氰呼吸降低,一方面导致花药物质代谢和能量代谢的紊乱,不育系花药不能利用药隔组织中的淀粉粒,另一方面不能有效将细胞内过多电子通过抗氰呼吸传至O₂,引致不育花药中O^-₂·升高,从而导致MDA含量在单核期和双核期均高于保持系,同时POD的活性在单核期及双核期均低于保持系,而SOD活性在单核期高于保持系,在双核期则低于保持系。不育系花药在发育中,花药O^-₂·和MDA过量积累,以及SOD和POD酶活性降低,导致活性氧产生与清除失去平衡,花粉败育。     

Flowering and morphological characterization of Dendrocalamus asper androecium and pollen grains

Abstract

Little is known about the reproduction of Dendrocalamus asper because it flowers only every 100 to 120 years. In the present work we describe some reproductive features of this bamboo and characterise flowers and pollen at various developmental stages. Number of pollen grains and ovules per flower, pollen/ovule ratio, in vitro twinning and pollen grain viability in vivo were evaluated and the different stages of floral development identified. Further, we performed a morphological analysis of androecium and pollen development. Seven distinct stages of flower development were identified; four initial stages, a pre-anthetic stage, and two stages of anthetic. Dendrocalamus asper pseudospikelets avoid inbreeding by means of protogyny. The floral and pollen characteristics suggest that the species is anemophilous. The ultrastructural characteristics of free microspores (stage two of floral development), vacuolated microspores (stage five) and mature pollen (anthetic) were analysed. During maturation, pollen grains accumulate larger and more numerous amyloplasts and organelles such as mitochondria. Pollen disperse in the tricellular development stage. Pollen is monoporate with an operculum-like pore, with a rugulate structure and a spinose tectum.     

大白菜新型细胞质雄性不育系6w-9605A的育性鉴定和花药败育的细胞学观察

采用石蜡切片技术,研究了大白菜(Brassica campestris L.ssp.pekinensis)细胞质雄性不育系6w-9605A及其保持系6w-9605B的花药发育过程的细胞形态学特征,确定不育系花药败育时期及方式,并对不育系6w-9605A进行花器官观察和育性鉴定.结果表明:保持系6w-9605B花药发育正常;不育系6w-9605A花药发育受阻于孢原分化时期,占总败育花药的66.7％,不形成花粉囊和花粉粒,属于无花粉囊型败育;另外33.3％的败育花药可形成花粉囊,小孢子均受阻于单核靠边期或者二胞期,败育特点为绒毡层细胞异常肥大,挤压小孢子,导致小孢子和绒毡层解体;6w-9605A的不育性稳定、彻底,不育株率和不育度均为100％.     

Involvement of phenylalanine ammonia-lyase in the development of pollen in broccoli (Brassica oleracea L.)

Summary To determine whether phenylalanine ammonia-lyase (EC 4.3.1.5) is involved in the maturation of microspores to fertile pollen, anthers of a fertile strain of broccoli (Brassica oleracea L.) were studied in a comparison with anthers of a cytoplasmic male sterile strain. In the normal fertile strain, immature anthers of about 2 mm in length exhibited higher phenylalanine ammonia-lyase activity than mature anthers or those shorter than 2 mm. The 2-mm-long anthers corresponded to the mononucleate stage, just after release of the microspores during pollen development. Immunohistochemical localization of phenylalanine ammonia-lyase in the anthers indicated that the protein was present predominantly in the tapetal cells. The immature anthers of cytoplasmic male sterile broccoli had a lower phenylalanine ammonia-lyase activity than those of the normal fertile strain. The level of phenylalanine ammonia-lyase activity in the immature anthers was positively correlated with the number of fertile pollen grains at the flowering stage in both strains. It seems possible, therefore, that phenylpropanoid metabolism, which involves phenylalanine ammonia-lyase, may play an important role in the maturation of microspores in flowering plants.Abbreviations CHS chalcone synthase - CMS cytoplasmic male sterility - DAPI 4, 6-diamidmo-2-phenylindole dihydrochloride - PAL L-phenylalanine ammonia-lyase     

Stage-related expression of mRNAs during pollen development in lily and tobacco

Homogeneous populations of developing microspores and pollen from anthers of lily (Lilium longiflorum Thumb.) and tobacco (Nicotiana tabacum L.) show a continuous production of biomass, reaching a maximum in young pollen. The rate of RNA synthesis was 460 fg · h^–1 in young binucleate cells, 138 fg · h^–1 in late binucleate cells and 56 fg · h^–1 in microspores. The mRNA population in developing pollen can be separated into three groups. In the first group, certain types of mRNAs are present at a constant level during all stages of development. A second group is characteristic of young pollen and increases quantitatively until anthesis. A third group is seen transiently; to this belong mRNAs present only before mitosis or at a distinct cell stage after mitosis. Some of the translation products of this latter group of mRNAs showed similarities between lily and tobacco on two-dimensional gels in respect of molecular weight and isolectric point, indicating that those mRNAs and proteins play a role in the regulation of pollen development.Abbreviations cDNA copy DNA - pI isolectric point To whom correspondence should be addressed.     

芝麻核雄性不育系ms86-1微粉发生的细胞学观察

芝麻(Sesamum indicum)核雄性不育系ms86-1姊妹交后代表现为可育、部分不育(即微粉)及完全不育(简称不育)3种类型。不同育性类型的花药及花粉粒形态差异明显。Alexander染色实验显示微粉植株花粉粒外壁为蓝绿色, 内部为不均一洋红色, 与可育株及不育株花粉粒的染色特征均不相同。为探明芝麻微粉发生机理, 在电子显微镜下比较观察了可育、微粉、不育类型的小孢子发育过程。结果表明, 可育株小孢子母细胞减数分裂时期代谢旺盛, 胞质中出现大量脂质小球; 四分体时期绒毡层细胞开始降解, 单核小孢子时期开始出现乌氏体, 成熟花粉时期花粉囊腔内及花粉粒周围分布着大量乌氏体, 花粉粒外壁有11–13个棱状凸起, 表面存在大量基粒棒, 形成紧密的覆盖层。不育株小孢子发育异常显现于减数分裂时期, 此时胞质中无脂质小球出现, 细胞壁开始积累胼胝质; 四分体时期绒毡层细胞未见降解; 单核小孢子时期无乌氏体出现; 成熟花粉时期花粉囊腔中未发现正常的乌氏体, 存在大量空瘪的败育小孢子, 外壁积累胼胝质, 缺乏基粒棒。微粉株小孢子在减数分裂时期可见胞质内有大量脂质小球, 四分体时期部分绒毡层发生变形, 单核小孢子时期有部分绒毡层开始降解; 绒毡层细胞降解滞后为少量发育进程迟缓的小孢子提供了营养物质, 部分小孢子发育为正常花粉粒; 这些花粉粒比较饱满, 表面有少量颗粒状突起, 但未能形成覆盖层, 花粉囊腔中及小孢子周围存在少量的乌氏体。小孢子形成的育性类型与绒毡层降解是否正常有关。     

Pollen wall and tapetum development in Plantago major L. (Plantaginaceae): assisting self-assembly

We have attempted to elucidate the underlying mechanisms of sporoderm development and pattern determination in Plantago major through a detailed ontogenetic study, using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). We aim to compare our observations and interpretation with those on other species. Our study of sporoderm development in Plantago from the early tetrad stage to mature pollen grains has shown that pure physical processes, including self-assembly, which are not under direct genetic control, play an important role and represent evidently one of the instruments of evolution. Our observations fit well with the sequence of self-assembling micellar mesophases and show reiteration of some of them, confirming our self-assembly hypothesis. Some attention was also paid to the possible role of rough and smooth endoplasmic reticulum in the cortical cytoplasm of the developing microspores. The tapetum and Ubisch bodies development are also traced. The importance of detailed ontogenetic studies for understanding the establishment of complex pollen walls in any species and for understanding mechanisms underlying sporoderm development was demonstrated. We also present a simulation, obtained in vitro experiments by self-assembly, mimicking pollen grain of Plantago major. It is clear that, in pollen wall development, biological processes and purely physical factors work in tandem.