Cytological Basis of Plastid Inheritance in Phaseolus vulgaris-Investigations of Plastids,Mitochondria and Their DNA Nucleoids During Pollen Development

Electron microscopic and DNA fluorescence microscopic observations of the plastids, mitochondria and their DNA in the developing pollen of Phaseolus vulgaris L. have demonstrated that the male plastids were excluded during microspore mitosis. The formed generative cell was free of plastids because of regional localization of plastids in early developing microspore and the extremely unequal distribution during division. The fluorescence observations of DNA showed that cytoplasmic (plastid and mitochondria) nucleoids degenerated and disappeared during the development of microspore/pollen, and were never presented in the generative cell at different development stages. These results provided precise cytological evidence of maternal plastid inheritance in Phaseolus vulgaris, which was not in accord with the biparental plastid inheritance identified from early genetic analysis. Based on authors' previous observations in a variety of common bean that the organelle DNA of male gamete was completely degenerated, the early genetic finding of the biparental plastid inheritance was unlikely to be effected by genotypic difference. Thus those biparental plastid inheritance might be caused by occational male plastid transmission, and plastid uniparental maternal inheritance was the species character of Phaseolus vulgaris.     

Embryological Studies of Paphiopedilum godefroyae Stein

P. godefroyae is one of the diandrous species of rather primitive orchids. The cytokinesis of PMCs conforms to simultaneous type. The arrangement of microspores in a tetrad is tetrahedral or isobilateral. The first mitosis in a pollen grain is unequal and results in the formation of two unequal cells. The small one is the generative cell and the large one, the vegetative cell. The wall material between them is callose which is easily detectable under the fluorescence microscope. When the generative cell detaches from the microspore wall and migrates into the cytoplasm of the vegetative cell, the callose wall disappears and a thin PAS-positive wall Was observed around the generative cell. The PAS-positive wall remains untill anthesis. The tapetum is of secretory type and its cells are binucleate. With the degradation of the tapetal cells, they discharge a lot of yellow, amorphous, sticky mass into the pollen sac. The pollens distribute in it to form a sticky pollen mass. The ovule has single integument and one layered nucellus around the magaspore mother cell. The mature embryo sac consists of eight or six cells and conforms to the Allium type. The interval between pollination and fertilization is about 45 days and the normal double fertilization has been observed. The primary endosperm cell undergoes one division only and results in the formation of 2 nucleate endosperm. The dormancy period of zygote lasts 45–50 days. During the development of the embryo, a suspensor consisting of a row of two to four cells is formed. It takes more than six months from the pollination to the maturation of the seed. The embryo in the mature seed is just an ellipsoidal mass of 120–140 cells without differentiation. The endosperm and suspensor are all degenerated in the mature seed.     

菜豆花粉发育中质体和线粒体及其DNA存在的状况——着重阐明质体遗传的细胞学基础

应用电镜和DNA的DAPI荧光检测技术研究了菜豆(Phaseolus vulgaris L.)小孢子/花粉发育中质体和线粒体及其DNA存在的状况。观察表明:在小孢子分裂时质体全部分配到营养细胞中,初形成的生殖细胞已不含质体。线粒体和质体的DNA在花粉发育中也先后降解,生殖细胞从刚形成时发育至成熟花粉时期这两种细胞器DNA均不存在。研究结果为菜豆质体母系遗传提供了确切的细胞学证据。遗传分析的研究曾确定菜豆质体为双亲遗传,对与本研究结论不同的原因进行了讨论。     

Unequal distribution of DNA-containing organelles in generative and sperm cells of Erythrina crista-galli (Fabaceae)

The generative cell at anthesis in the mature pollen grain of Erythrina crista-galli (Fabaceae) was examined by 4,6-diamidino-2-phenylindole(DAPI)-fluorescence microscopy using the squash method. An unequal, polarized distribution of DNA-containing organelles (plastids and/or mitochondria) within the generative cell was observed in every mature pollen grain examined. Polarization of DNA-containing organelles is obvious when generative cells are freed and assume a spherical shape soon after microspore mitosis, as revealed by fluorescence-microscopic observations of specimens embedded in Technovit 7100 resin and thin-sectioned at different developmental stages. Early establishment of polarized localization of organelles in young generative cells of E. crista-galli and maintenance of this unequal distribution until pollen maturation strongly suggests that the organelles may still be clustered at pollen mitosis. Production of a dimorphic pair of sperm cells, as has been reported in Plumbago zeylanica, was observed in some pollen tubes germinated in vitro. The differentiation of the two sperm cells is discussed in relation to possible preferential double fertilization in angiosperms. Received: 28 July 1999 / Revision accepted: 8 November 1999     

Detection and quantification of rRNA by high-resolutionin situ hybridization in pollen grains

We examined changes in the localization of cytoplasmic rRNA during pollen development inNicotiana tabacum SR-1. The rRNA was visualized byin situ hybridization, and the signal intensity of rRNA in microspore, vegetative and generative cell was quantified by microphotometry. The amount of rRNA per microspore or pollen section increased about 5 times from microspore to mature pollen grain and kept increasing even in the late stage of pollen development after PMI. The increase of rRNA occur in both vegetative and generative cells. The results suggest that synthesis of rRNA occur even after PM I in both vegetative and generative cells.     

Ultrastructural and Cytochemical Study on Mature Pollen of Pinus tabulaeformis

The pollen of Pinus tabulaeformis Cart. comprised two prothallial cells, a generative cell and a tube cell which degenerated at pollen maturation. The generative cell had its own cell wall, seperating from the intine of pollen, but with its side wall attached to the infine. Cytoplasmic channels were present on the side of the generative cell wall, which faced to the tube cell cytoplasm. The generative cell differed conspicuously from the tube cell. The main differences include: ( 1 ) The chromatin in the generative cell nucleus was condensed, but was dispersed and had numerous nueleare pores in the tube cell nucleus; (2)There was no microbody in the generative cell but many microbodies were present in the tube cell cytoplasm; (3)More inclusions were present in the tube cell than in the generative cell. Both the generative cell and the tube cells contained lipid bodies and amyloplasts in the cytoplasm, but there were more amyloplasts in the former. The tube cell also contained a few proteins which was absent in the generative cell. In addition, there were numerous mitochondria, polyribosomes, and a few endoplasmic reticulums and dictyosomes in the generative and tube cells. DAPI staining demonstrated numerous cytoplasmic DNA in both generative cell and tube cell. The mode of cytoplasmic inheritance, and the composition, structure and the nature of the pollen wall of P. tabulaefonnis are also discussed in this paper.     

Microsporogenesis and microgametogenesis of the Argentinian species of Podocarpus (Podocarpaceae)

The development of the microsporangium and male gametophyte of three species of Podocarpus was studied with light microscopy (LM) and the morphology of pollen with scanning and transmission electron microscopy (SEM and TEM). During early stages, the male cone is covered with coriaceous scales. The archesporid cells go through a dormant period. Later the pollen mother cells differentiate and undergo meiosis. Callose is detected around the tetrad and between each monad. The microspore nucleus divides several times to give rise to a multicellular gametophyte, which includes the tube cell, the stalk and body cells, and four prothallial cells. The exine of the pollen grain is rugulate in the corpus and quite smooth in the sacci. The ultrastructure of the pollen wall consists of the alveolate sexine, the laminate nexine I and the amorphous nexine II. The intine is very thin. Comparison of the mature grain of some fossils with living members of the Podocarpaceae reveals great similarity.     

Arabidopsis CDKA;1, a cdc2 homologue, controls proliferation of generative cells in male gametogenesis

The protein kinase cdc2 is conserved throughout eukaryotes and acts as a key regulator of the cell cycle. In plants, A-type cyclin-dependent kinase (CDKA), a homologue of cdc2, has a role throughout the cell cycle. Here we show that a loss-of-function mutation in CDKA;1, encoding the only Arabidopsis CDKA, results in lethality of the male gametophyte. Heterozygous plants produced mature siliques containing about 50% aborted seeds, and segregation distortion was observed in paternal inheritance. Microspores normally undergo an asymmetric cell division, pollen mitosis I (PMI), to produce bicellular pollen grains. The larger vegetative cell does not divide, but the smaller generative cell undergoes mitosis, PMII, to form the two sperm cells, thereby generating tricellular pollen grains. The cdka-1 mutant, however, produces mature bicellular pollen grains, consisting of a single sperm-like cell and a vegetative cell, due to failure of PMII. The mutant sperm-like cell is fertile, and preferentially fuses with the egg cell to initiate embryogenesis. As the central cell nucleus remains unfertilized, however, double fertilization does not occur. In heterozygous plants, the embryo is arrested at the globular stage, most likely because of loss of endosperm development, whereas it is arrested at the one- or two-cell stage in presumptive homozygous plants. Thus, CDKA;1 is essential for cell division of the generative cell in male gametogenesis.     

Development of male gametes in flowering plants

The male gametes of angiosperms consist of two sperm cells within a pollen grain or a pollen tube. They are derived from a single generative cell, which is formed as the smaller cell by unequal cell division in the microspore after meiosis. Limited information is available about these male gametic cells, beyond observations by electron microscopy, because each is surrounded by the cytoplasm of a larger vegetative cell. Recently, large quantities of generative cells and sperm cells have been isolated from pollen grains or pollen tubes of various plant species, and their physiological, biochemical and molecular characterization is now possible. Although almost all the available results are still preliminary, it is evident that the male gametic cells are peculiar in terms both of cell structure and composition. For example, they are rich in axial microtubules which maintain the spindle-like shape of each cell. However, they lack plastids which are DNA-containing cytoplasmic organelles. Biochemical characterization of their proteins indicates the presence of male gamete-specific polypeptides. These findings suggest, not unexpectedly, the possibility of male gamete-specific gene expression and of a strict genetic mechanism that controls the formation of male gametes.     

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。     

新型小麦胞质不育系花粉败育的细胞学观察

观察了１种新型小麦细胞质雄性不育素（ＣＭＳ）－（野生二粒小麦）中国春ＣＭＳ花药和花粉败育的细胞学过程,结果表明;（１）不育系在小孢子发育至单核晚期以前,除了雄蕊心皮化发生率（３７．２％）较高外,其花药和花粉发育绝大多数与同核保持系相似,是正常的,仅少量表现异常而导致败育,异常现象主要有：雄蕊心皮化。药室合并,药壁组织喙状突起,绒毡层异常,小孢子母细胞粘连,减数分裂异常,小孢子异常等。（２）不育系花     

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     

La glutamate déshydrogénase au cours de la gamétogénèse mâle chez Medicago sativa

Glutamate dehydrogenase during male gametogenesis in Medicago sativa.
The multiple forms of the alfalfa ( Medicago sativa L.) glutamate dehydrogenase (GDH; EC 1.4.1.2–4) are much more numerous in the pollen than in other organs, and in particular in the floral parts. The appearance of the pollen-specific GDH pattern during male gametogenesis has been followed using electrophoretic, cytological and immunochemical techniques. After the tetrad stage the uninucleate microspore has no glutamate dehydrogenase activity. This activity appears only in the mitochondria of the binucleate young pollen. At this stage the electrophoretic pattern displays a faint intensity, but is already identical to that of mature pollen. On the other hand, the use of monospecific antibodies has revealed the presence of antigen in the microspore, and in the young pollen there is as much antigen as in the mature pollen. Therefore the synthesis of GDH precedes its activation, which follows the first pollen mitosis.     

莴苣花粉发育过程中ATP酶的分布

对莴苣花粉发育过程中ATPase的分布特征做了研究。四分体早期的小孢子细胞质中开始出现ATPase反应颗粒。之后,小孢子在发育过程中,花粉内壁聚集大量体积较大的ATPase反应颗粒,并一直保持到花粉即将成熟。在小孢子发育晚期,在花粉萌发孔处和小孢子大液泡中也特异性地聚集了较多ATPase颗粒。二胞花粉刚形成的生殖细胞表面呈现大量的ATPase反应颗粒,当生殖细胞脱离花粉内壁移入营养细胞,ATPase反应颗粒基本消失。生殖细胞分化过程中生殖细胞的ATPase反应颗粒逐渐低于营养细胞中的。在成熟花粉中,精细胞中的ATPase反应颗粒比营养细胞中的少,且主要集中在细胞核中。结果显示花粉发育过程中ATPase的特异分布与花粉发育的一些生物学事件密切相关。     

MICROGAMETOPHYTE DEVELOPMENT IN THE PALEOZOIC SEED FERN FAMILY CALLISTOPHYTACEAE

Well-preserved stages of microgametophyte development are described from pollen produced by the Paleozoic seed fern family Callistophytaceae. Microgametophyte development in both the Middle Pennsylvanian pollen organ Idanothekion and Upper Pennsylvanian Callandrium involved the initial production of an axial row of at least three small prothallial cells proximally and a large embryonal cell distally. The arrangement and form of these cells is like that present in some extant genera of the Pinaceae. The prothallial cells were relatively large in comparison with extant gymnosperms, occupying the entire region of the cap-pus, and were apparently all primary. Evidence is presented that in Callandrium further development involved an anticlinal division of the large distal cell (antheridial initial) into a small generative cell contained within a larger tube cell. Previously described microgametophytes of the late Paleozoic order Cordaitales are reinterpreted and are shown to consist of an embryonal cell and three to four discoidal prothallial cells in an axial row like that of the Callistophytaceae. Microgametophytes thus far described from the Paleozoic are remarkably modern in appearance and provide no evidence to support the generally held view that the seed plant microgametophyte is an extremely reduced sexual phase that has arisen through the loss of almost all of the vegetative cells and the sterile outer cells of the antheridium. Evidence to support or refute this view will depend upon the discovery of microgametophytes from older groups of seed plants than those for which they are now known.     

芡实绒毡层细胞发育的超微结构变化

芡实（ Ｅｕｒｙａｌｅｆｅｒｏｘ Ｓａｌｉｓｂ） 绒毡层细胞在小孢子母细胞时期, 质体出现明显的变形期,细胞中二核常相互贴近或呈嵌合状态, 细胞壁间层中胞间连丝发达。减数分裂期, 绒毡层细胞壁融解消失, 胞间连丝断离, 细胞间发育出现不同步现象。质体开始积累淀粉, 部分质体呈空泡状, 并出现质体膜内陷, 这与液泡具相似的功能。四分体时期, 绒毡层细胞内部结构开始解体。单核小孢子时期, 绒毡层细胞解体消失, 使小孢子后期发育的营养来源受到影响,作者认为这是生产上成熟花粉囊中花粉粒少而且发育不正常的主要原因之一。     

Microsporogenesis inPinus sylvestris L. VIII. Tapetal and late pollen grain development

This last portion of our developmental study ofPinus sylvestris L. pollen grains extends from just prior to the first microspore mitosis to the microsporangial dehiscence preparatory to pollen shedding. In nine years of collecting each day the duration of the above period was 7 to 11 days. Tapetal cells extended into the loculus and embraced microspores during the initial part of the above period. Thereafter tapetal cells receded, became parallel to parietal cells and so imbricated that there appeared to be two or three layers of tapetal cells. Tapetal cells were present up to the day before pollen shedding, but only rER and some mitochondria appeared to be in good condition at that time. A callosic layer (outer intine) was initiated under the endexine before microspore mitosis. After the first mitosis the first prothallial cell migrated to the proximal wall and was covered on the side next to the pollen cytoplasm by a thin wall joining the thick outer intine. There are plasmodesmata between pollen cytoplasm and the prothallial cell. After the second mitosis the second prothallial cell became enveloped by the outer intine. The inner intine appears after formation of the two prothallial cells but before the third mitosis. During this two-prothallial cell period before the third mitosis, plastids had large and complex fibrillar assemblies shown to be modified starch grains. After the third mitosis plastids of the pollen cytoplasm contained starch and the generative cell (antheridial initial), the product of that mitosis, is enveloped by the inner intine. On the day of pollen shedding cells are removed from the microsporangial wall by what appears to be focal autolysis. The tapetal and endothecial cells for 10–15 µm on each side of the dehiscence slit are completely removed. One or more epidermal cells are lysed, but both a thin cuticle and the very thin sporopollenin-containing peritapetal membrane remain attached to the undamaged epidermal cells bordering the dehiscence slit. Our study terminates on the day of pollen shedding with mature pollen still within the open microsporangium. At that time there is no longer a clear morphological distinction between the outer and inner intine but, judging by stain reactions, there is a chemical difference. The exine of shed pollen grains was found to be covered by small spinules on the inner surface of alveoli. These had the same spacing as the Sporopollenin Acceptor Particles (SAPs) associated with exine initiation and growth.     

白皮松花粉发育过程中蛋白酶体活性及其分布的动态变化

蛋白酶体途径对花粉发育调控具有重要作用, 但花粉发育过程中蛋白酶体的分布及其活性的动态变化一直未见报道。蛋白酶体荧光底物结合荧光分光光度计分析表明, 蛋白酶体的活性从单核小孢子到具有2个原叶细胞的三细胞花粉逐渐增强, 而在成熟花粉中略有下降。免疫荧光标记结合共聚焦显微镜分析表明, 蛋白酶体不均匀地分布于细胞质和细胞核中, 并在花粉细胞不均等分裂过程中聚集分布于先后产生的2个原叶细胞内。总之, 蛋白酶体的活性及其分布在花粉发育过程中存在相关的时空动态变化, 表明裸子植物花粉中的蛋白酶体活性及其分布与花粉发育具有相关性, 并在原叶细胞的退化过程中起重要作用。     

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.