Isolation and characterization of neutral-lipid-containing organelles and globuli-filled plastids from Brassica napus tapetum

The monolayer tapetum cells of the maturing flowers of Brassica napus contain abundant subcellular globuli-filled plastids and special lipid particles, both enriched with lipids that are supposed to be discharged and deposited onto the surface of adjacent maturing pollen. We separated the two organelles by flotation density gradient centrifugation and identified them by electron microscopy. The globuli-filled plastids had a morphology similar to those described in other plant species and tissues. They had an equilibrium density of 1.02 g/cm3 and contained neutral esters and unique polypeptides. The lipid particles contained patches of osmiophilic materials situated among densely packed vesicles and did not have an enclosing membrane. They exhibited osmotic properties, presumably exerted by the individual vesicles. They had an equilibrium density of 1.05 g/cm3 and possessed triacylglycerols and unique polypeptides. Several of these polypeptides were identified, by their N-terminal sequences or antibody cross-reactivity, as oleosins, proteins known to be associated with seed storage oil bodies. The morphological and biochemical characteristics of the lipid particles indicate that they are novel organelles in eukaryotes that have not been previously isolated and studied. After lysis of the tapetum cells at a late stage of floral development, only the major plastid neutral ester was recovered, whereas the other abundant lipids and proteins of the two tapetum organelles were present in fragmented forms or absent on the pollen surface.

水稻花药绒毡层特异基因及减数分裂相关基因的分离与功能分析

　利用RNA减法杂交、差异筛选和5’-RACE等方法从水稻分离到了一花药绒毡层特异表达的基因RA39。Southern 杂交表明，RA39在水稻基因组中是以单拷贝的形式存在的。RT-PCR 结果初步表明，RA39是一水稻花药特异表达的基因。RNA原位杂交进一步表明，RA39主要在水稻花药的绒毡层中表达，而且在小孢子母细胞减数分裂期和四分体时期表达量最高。RA39 cDNA全长1013bp，编码298个氨基酸残基。 RA39 cDNA与数据库中的已知序列没有明显的相似性，由其推测的多肽与核糖体失活蛋白（ribosome-inactivating protein, RIP）的序列相似在19-34%之间。多重序列排列分析结果表明构成RIPs活性位点的5个关键氨基酸残基在RA39中是保守的，在蓖麻毒蛋白中分别为Tyr80、 Tyr123、 Glu177、 Arg180 and Trp211 。利用原核表达系统，通过蛋白质分离和纯化获得了在SDS电泳图谱上为单一条带的纯的RA39蛋白，用兔rRNA作底物进行的酶活性分析证明该蛋白有N-糖基化作用，是一种类型I的核糖体失活蛋白。反义转基因植株的花粉用TTC进行活性染色结果显示其活性明显减弱，成熟的T0代反义转基因植株的结实率明显降低，只有对照的20-60%。这说明，RA39蛋白可能和小孢子母细胞的发育相关。 　　酵母DMC1是减数分裂过程中同源染色体配对和重组修复所必需的减数分裂特异基因。根据酵母Dmc1和拟南芥AtDmc1的保守区设计简并性引物，通过RT-PCR和RACE等方法，从水稻中分离出了酵母DMC1的同源基因OsDMC1。RT-PCR分析表明，OsDMC1在花中表达量最高，在根中表达量较低，在叶片和幼芽几乎不表达。水稻基因组中有两个拷贝的OsDMC1。OsDmc1蛋白与酵母Dmc1和拟南芥AtDmc1氨基酸一致性分别为53%和81%。 　　酵母Spo11在减数分裂过程中具有催化DNA双链断裂从而起始同源重组的功能。以酵母Spo11氨基酸序列为探针和现有的数据库通过数据分析，结合RACE技术，克隆了水稻SPO11同源基因OsSPO11-1， OsSPO11-1是一个单拷贝基因，有3个外显子和2个内含子，在转录过程中通过内含子的可变剪切产生4个不同的转录本（OsSPO11-1A、OsSPO11-1B、OsSPO11-1C和OsSPO11-1），其中，OsSPO11-1A是一个未剪切的转录本，OsSPO11-1B包含内含子2，OsSPO11-1C包含内含子1，OsSPO11-1D是一个完全剪切的转录本。这些转录本编码的蛋白有一致的246氨基酸残基的C-端，包含了Spo11/TopVIA家族蛋白共有的5个功能基元，是该家族的新成员。OsSPO11-1A和 OsSPO11-1C在花中优势积累，OsSPO11-1B是花特异的，而OsSPO11-1D在营养器官中优势积累。在花中该基因主要在减数分裂的花粉母细胞和胚曩中表达，在减数分裂期的绒毡层细胞和不同花器官的微管束细胞中也表达。这些结果说明内含子涉及到了OsSPO11-1表达的器官特异性调节，该基因除了参与减数分裂的调节外，在体细胞的发育中可能起重要作用。

Embryology of Swertia cincta (Gentianaceae) and Its Systematic Value

This paper reports the mega-, micro-sporogenesis and female-, male-gametogenesis of Swertia cincta for the first time, with the aim of discussing the systematic position of section Platynema and section Ophelia of Swertia. Anthers are tetrasporangiate. The development of anther walls conforms to the dicotyledonous type. The tapetum cells have dual origin and are similar to the glandular type. There are two middle layers. The endothecium and epidermis persist. Cytokinesis in the microsporrocyte meiosis is simultaneous type and the microscpore teads are tetrahedral. Pollen grains are 3-celled. The ovary is bicarpellum and unilocular. The placentation is of suparietal placentation with 12 series of ovules. The ovules. The ovule is unitegmic, tenuinucellar and ana-campylotropous, The embryo sac orignates from the single-archesporial cell. The one chalazal megaspore in lienar tetrad become the functional megasore. The development of embryo sac is of the polygonum type. Before fertilization, two polar nuclei fuse into one secondary nucleus. Three antipodal cells persisted and divided into 5-8 cells. A comparison between two sections indicates that section Plathnema is better treated as distinct section and is more advanced than section Ophelia according to the evolutionary trends of embryological characters.

Embryology of Swertia (Gentianaceae) relative to taxonomy

The embryological features of three species of Swertia (s.l.) - S. erythrosticta, S. franchetiana, and S. tetraptera were characterized, and the observations were used, together with previously gathered data on other species, to evaluate a recently proposed polyphyly, based on molecular data, of Swertia s.l. Comparisons of species within the genus showed that they have diversified embryologically, and there are significant between-species differences. Notable features that vary between species include the number of cell layers that form the anther locule wall, the construction of the wall of the mature anther, tapetum origin, the cell number in mature pollen grains, the structure of the fused margins of the two carpels, the ovule numbers in placental cross-sections, the shape of the mature embryo sac, the degree of ovule curvature, antipodal variation and the presence of a hypostase, and seed appendages. They share characters that are widely distributed in the tribe Gentianeae, such as a dicotyledonous type of anther wall formation, a glandular tapetum with uninucleate cells, simultaneous cytokinesis following the meiosis of the microsporocytes, tetrahedral microspore tetrads, superior, bicarpellary and unilocular ovaries, unitegmic and tenuinucellar ovules, Polygonum-type megagametophytes, progamous fertilization, nuclear endosperm, and Solanad-type embryogeny. The presence of variation in embryological characters amongst the species of Swertia s.l. strongly supports the view that Swertia s.l. is not a monophyletic group. Frasera is better separated from Swertia s.l. as an independent genus, and is only distantly related to Swertia s. s. judging from the numerous differences in embryology. Swertia tetraptera is very closely related to Halenia, as they show identical embryology. (C) 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 155, 383-400.

Embryology of Crawfurdia delavayi (Gentianaceae) and its systematic value

The embryological characters of Crawfurdia delavayi Frabnch. are described and the systematic relationships of Crawfurdia discussed. Anthers are tetrasporangiate. The development of anther walls conforms to the Dicotyledonous type. The tapetum is of single origin. The development of the tapetum with uninucleate cells is of the glandular type. The tapetal cells on the connective side show radial elongation or periclinal division and intrude into the anther locule. The epidermis of anther walls persists and its cells become pillar and fibrous, and the endothecium degenerates. The ovary is bicarpellary and unilocular. The placentation is typically parietal with 8 rows of anatropous ovules. The development of embryo sac is of the polygonum type. Before fertilization, two polar nuclei fuse into a secondary nucleus. Three antipodal cells persist. Flowers are protandrous. Fertilization is porogamous. The development of the endosperm is of the nuclear type. The embryogeny corresponds to the solanad type physalis II variation. The embryological data indicate that it is better to separate Crawfurdia from Gentiana as an independent genus.

Microsporogenesis and anther development in Bromeliaceae

Bromeliaceae possess several features of pollen and anther wall development that are plesiomorphic for Poales, consistent with their putatively basal or near-basal placement in this order. For example, successive microsporogenesis and the monocotyledonous type of anther wall formation are both plesiomorphic features that occur commonly in other Poales, with a few notable exceptions, notably the simultaneous type of microsporogenesis in Rapateaceae. The intermediate type of tapetum development in Bromeliaceae was probably derived secondarily from a secretory type, which occurs in most other Poales except Typhaceae.

Anther development, microsporogenesis and microgametogenesis in Heliconia (Heliconiaceae, Zingiberales)

Anther development, micro sporogenesis and microgametogenesis in several species of Heliconia were investigated as part of a complementary embryological study of the Heliconiaceae. All studied Heliconia species present bithecate and tetrasporangiate anthers with fertile pollen grains; only H. rivularis, a natural hybrid, presented sterile pollen grains of variable size and no content. The anther wall has an uniseriate epidermis and endothecium, the latter with helicoidal thickenings, although some cells of the middle layers also showed thickenings; the biseriate tapetum is of amoeboid non-syncytial type, since the tapetum cells did not fuse together forming a true plasmodium. The microsporogenesis is successive leading to isobilateral tetrads. The inaperturate pollen grains had a very reduced exine consisting of a thin, more or less continuous layer with small spines upon; the pollen grain shape is variable among the species, all of them presenting heteropolar pollen, except H. angusta with isopolar ones. Most of these characteristics were shared with other studied Zingiberales, although more studies need to be done. (c) 2006 Elsevier GmbH. All rights reserved.

Embryology and seed development of Blastocaulon scirpeum and Paepalanthus scleranthus (Eriocaulaceae)

The embryology and seed structure of Blastocaulon scirpeum (Mart.) Giul. and Paepalanthus scleranthus Ruhland were studied in order to contribute to the embryology of Eriocaulaceae and supply data for future taxonomic studies. Both species present: anther with 4-layered wall; conspicuous endothecium with fibrous thickenings; secretory tapetum with uninucleate cells; successive microsporogenesis forming isobilateral microspore tetrads; bicellular pollen grains; orthotropous, bitegmic and tenuinucellate ovule; micropyle formed by the inner integument alone; megagametophyte of the Polygonum type, with a conspicuous antipodal cyst; nuclear and starchy endosperm; reduced, undifferentiated, and bell-shaped embryo; operculate and endotestal seed; seed coat derived from the two ovule integuments; and tanniniferous endotegmen. In addition, Blastocaulon scirpeum shows a bisporangiate anther and a 3-layered ovary wall, while P. scleranthus presents a tetrasporangiate anther that becomes bisporangiate at maturity, and a 2-layered ovary wall. This investigation shows that the bisporangiate condition does not suffice to separate Blastocaulon from Paepalanthus, since it is common to both. It also indicates, based on several embryological aspects, the proximity of Eriocaulaceae and Xyridaceae, which comply mainly with the features presented by the other commelinid families. These results may be used in future cladistic analysis of the family, and contribute to a better understanding of its phylogeny.

Anther and pollen development in some species of Poaceae (Poales)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Flower morpho-anatomy in Epiphyllum phyllanthus (Cactaceae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Morphology and development of anthers and ovules in Croton and Astraea (Euphorbiaceae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Embryology and seed development of Syngonanthus caulescens (Poir.) Ruhland (Eriocaulaceae-Poales)

The embryology and the seed development of Syngonanthus caulescens are presented. This species possesses: a bithecous and tetrasporangiate anther, with a four-layered wall, a conspicuous endothecium of the baseplate type, a secretory tapetum formed by uninucleate cells, successive microsporogenesis resulting in isobilateral microspore tetrads, spiraperturate and binucleate pollen grains, an orthotropous, pendulous, bitegmic and terminucellate ovule, with a micropyle formed only by the inner integument, a megagametophyte of the Polygonum type, with formation of an antipodal cyst, free-nuclear and starchy endosperm, a broad and bell-shaped embryo, operculate and endotestal seeds, a seed coat derived from the inner layers of both integuments, and tanniniferous endotegmen. These embryological aspects are characteristic not only for Syngonanthus, but for the whole family, with few differences between genera. Furthermore, the pollen grain of the spiraperturate type and the cystic arrangement of the antipodals in the megagametophyte are peculiar and very distinctive features of Eriocaulaceae within the other Poales (commelinids). (c) 2006 Elsevier B.V. All rights reserved.

Development and cytology of pollen in Dahlstedtia Malme (Leguminosae : Papilionoideae)

Microsporogenesis, chromosome number, meiotic behaviour and meiotic index were investigated in Dahlstedtia pinnata and D. pentaphylla, two legume species occurring largely in Brazil, in order to ascertain whether the pollen could limit fertilization events. Archesporial cells originate primary sporogenous and anther wall precursor cells, the tapetum is uniseriate, uninucleate and glandular. Tetrads are tetrahedric or decussate, and cytokinesis is of the simultaneous type. Mature pollen grains are tricolpate and bicellular. No abnormalities in microsporogenesis were found. In both species the chromosome number is n = 11, a number not reported previously. The base number for Dahlstedtia is also 11, because cytological observations include both species of Dahlstedtia. D. pentaphylla has a higher meiotic index and lower individual variation values, and it is considered meiotically stable. Its pollen grains do not limit fertilization. D. pinnata has a lower meiotic index, and the pollen is one of the factors which limit fertilization. Furthermore, D. pinnata has numerous adventitious shoots, which suggest that vegetative propagation is important in its reproductive process. (C) 2002 the Linnean Society of London, Botanical Journal of the Linnean Society, 2002, 138, 461-471.