NUCLEAR FUSION DEFECTIVE1 encodes the Arabidopsis RPL21M protein and is required for karyogamy during female gametophyte development and fertilization

Karyogamy, or nuclear fusion, is essential for sexual reproduction. In angiosperms, karyogamy occurs three times: twice during double fertilization of the egg cell and the central cell and once during female gametophyte development when the two polar nuclei fuse to form the diploid central cell nucleus. The molecular mechanisms controlling karyogamy are poorly understood. We have identified nine female gametophyte mutants in Arabidopsis (Arabidopsis thaliana), nuclear fusion defective1 (nfd1) to nfd9, that are defective in fusion of the polar nuclei. In the nfd1 to nfd6 mutants, failure of fusion of the polar nuclei is the only defect detected during megagametogenesis. nfd1 is also affected in karyogamy during double fertilization. Using transmission electron microscopy, we showed that nfd1 nuclei fail to undergo fusion of the outer nuclear membranes. nfd1 contains a T-DNA insertion in RPL21M that is predicted to encode the mitochondrial 50S ribosomal subunit L21, and a wild-type copy of this gene rescues the mutant phenotype. Consistent with the predicted function of this gene, an NFD1-green fluorescent protein fusion protein localizes to mitochondria and the NFD1/RPL21M gene is expressed throughout the plant. The nfd3, nfd4, nfd5, and nfd6 mutants also contain T-DNA insertions in genes predicted to encode proteins that localize to mitochondria, suggesting a role for this organelle in nuclear fusion.     

Somatic small RNA pathways promote the mitotic events of megagametogenesis during female reproductive development in Arabidopsis

Female gamete development in Arabidopsis ovules comprises two phases. During megasporogenesis, a somatic ovule cell differentiates into a megaspore mother cell and undergoes meiosis to produce four haploid megaspores, three of which degrade. The surviving functional megaspore participates in megagametogenesis, undergoing syncytial mitosis and cellular differentiation to produce a multicellular female gametophyte containing the egg and central cell, progenitors of the embryo and endosperm of the seed. The transition between megasporogenesis and megagametogenesis is poorly characterised, partly owing to the inaccessibility of reproductive cells within the ovule. Here, laser capture microdissection was used to identify genes expressed in and/or around developing megaspores during the transition to megagametogenesis. ARGONAUTE5 (AGO5), a putative effector of small RNA (sRNA) silencing pathways, was found to be expressed around reproductive cells during megasporogenesis, and a novel semi-dominant ago5-4 insertion allele showed defects in the initiation of megagametogenesis. Expression of a viral RNAi suppressor, P1/Hc-Pro, driven by the WUSCHEL and AGO5 promoters in somatic cells flanking the megaspores resulted in a similar phenotype. This indicates that sRNA-dependent pathways acting in somatic ovule tissues promote the initiation of megagametogenesis in the functional megaspore. Notably, these pathways are independent of AGO9, which functions in somatic epidermal ovule cells to inhibit the formation of multiple megaspore-like cells. Therefore, one somatic sRNA pathway involving AGO9 restricts reproductive development to the functional megaspore and a second pathway, inhibited by ago5-4 and P1/Hc-Pro, promotes megagametogenesis.     

矮沙冬青雌配子体及胚胎发育研究

矮沙冬青子房单心皮1室,边缘胎座,弯生胚珠,胚珠具双珠被、厚珠心。大孢子孢原细胞发生于珠心表皮下,大孢子母细胞减数分裂形成直线排列的四分体,合点端大孢子具功能,并按蓼型胚囊发育,雌配子体成熟于4月中旬。双受精后,胚乳发育为核型。在矮沙冬青大孢子发生、雌配子体和胚胎发育过程中未发现异常现象,因此认为矮沙冬青濒危不存在雌性生殖结构与发育过程异常的内在因素。     

MAA3 (MAGATAMA3) helicase gene is required for female gametophyte development and pollen tube guidance in Arabidopsis thaliana

The female gametophyte plays a central role in the sexual reproduction of angiosperms. We previously isolated the maa3 (magatama3) mutant of Arabidopsis thaliana, defective in development of the female gametophyte, micropylar pollen tube guidance, and preventing the attraction of multiple pollen tubes. We here observed that the nucleolus of polar nuclei is small, and that the fusion of polar nuclei often did not occur at the time of pollination. The MAA3 gene encodes a homolog of yeast Sen1 helicase, required for RNA metabolism. It is suggested that MAA3 may regulate RNA molecules responsible for nucleolar organization and pollen tube guidance.     

八角莲大孢子发生和雌配子体形成

首次报道了八角莲(Dysosma versipellis (Hance)M.cheng)大孢子发生和雌配子体形成的过程.结果:双珠被,多为厚珠心胚珠,少数为假厚珠心,胚珠多为横生,少数为弯生;边缘胎座,子房一室,多胚珠,珠孔由两层珠被共同形成,呈"之"字形;多为单孢原,位于珠心表皮下:偶见2～3个孢原细胞位于珠心表皮下;大孢子母细胞有两种发生方式;直线形大孢子四分体,合点端的大孢子发育为功能大孢子,蓼型胚囊;成熟胚囊中,二个极核在受精前合并为次生核;三个反足细胞不发达,较早退化;"品"字形卵器极性明显,其中卵细胞与助细胞极性相反;助细胞发达,其丝状器在不同发育时期形态及大小不同,且具吸器功能.     

THE FEMALE GAMETOPHYTE OF MACHAERANTHERA PATTERSONII (ASTER PATTERSONII) AND OF M. TANACETIFOLIA

The nucellus of Machaeranthera pattersonii (A. Gray) Greene (Aster pattersonii A. Gray) contains only one megaspore mother cell, and the female gametophyte develops from the chalazal megaspore of a row of four, thus conforming to the Polygonum type of development. These observations are contrary to the older work of Palm. Three nuclear divisions produce the typical eight nuclei with the egg apparatus, primary endosperm cell with two polar nuclei, and two antipodal cells, the micropylar one containing two nuclei. Usually no more antipodal cells are formed, although there is further nuclear division, apparently followed by nuclear fusion. The antipodal cells remain about the same size without forming an antipodal haustorium. Cell division accompanies the first division of the primary endosperm nucleus. The early stages of the embryo resemble those of other Compositae. Machaeranthera tanacetifolia (HBK) Nees also shows the Polygonum type of development of the female gametophyte. It is suggested that Palm may have been working on some species of Erigeron that had been wrongly identified, which would account for the difference in observations.     

车桑子大孢子发生与雌配子体发育

采用常规石蜡切片法,对车桑子大孢子的发生和雌配子体的发育进行观察,探讨车桑子自然结籽率低的原因和明确其胚胎发育特征。结果表明:(1)车桑子花柱有花柱道,子房3室,中轴胎座,横生胚珠,每心室两枚胚珠,双珠被,厚珠心,无承珠盘。(2)位于珠心表皮细胞下的孢原细胞经平周分裂产生造孢细胞,造孢细胞发育为大孢子母细胞,大孢子母细胞经减数分裂形成线性四分体,靠近珠孔端3个大孢子退化消失,靠合点端大孢子发育为功能大孢子,大孢子发生类型为单孢子发生型。(3)单核胚囊经3次有丝分裂形成7细胞8核的成熟胚囊,胚囊发育类型为蓼型。(4)花器官形态的变化和大孢子发育过程有一定联系,可根据雌花形态特征大致判断大孢子发育时期。研究认为,车桑子雌配子体发育过程中出现的胚囊不中空、游离核不进一步细胞化等异常现象,可能是导致车桑子自然结籽率低的原因之一。     

The dyad gene is required for progression through female meiosis in Arabidopsis

In higher plants the gametophyte consists of a gamete in association with a small number of haploid cells, specialized for sexual reproduction. The female gametophyte or embryo sac, is contained within the ovule and develops from a single cell, the megaspore which is formed by meiosis of the megaspore mother cell. The dyad mutant of Arabidopsis, described herein, represents a novel class among female sterile mutants in plants. dyad ovules contain two large cells in place of an embryo sac. The two cells represent the products of a single division of the megaspore mother cell followed by an arrest in further development of the megaspore. We addressed the question of whether the division of the megaspore mother cell in the mutant was meiotic or mitotic by examining the expression of two markers that are normally expressed in the megaspore mother cell during meiosis. Our observations indicate that in dyad, the megaspore mother cell enters but fails to complete meiosis, arresting at the end of meiosis 1 in the majority of ovules. This was corroborated by a direct observation of chromosome segregation during division of the megaspore mother cell, showing that the division is a reductional and not an equational one. In a minority of dyad ovules, the megaspore mother cell does not divide. Pollen development and male fertility in the mutant is normal, as is the rest of the ovule that surrounds the female gametophyte. The embryo sac is also shown to have an influence on the nucellus in wild type. The dyad mutation therefore specifically affects a function that is required in the female germ cell precursor for meiosis. The identification and analysis of mutants specifically affecting female meiosis is an initial step in understanding the molecular mechanisms underlying early events in the pathway of female reproductive development.     

云南松雌雄配子体的发育

云南松(Pinus yunnanensis Fr.)雄配子体于10月在小孢子叶腹面产生二个小孢子囊,内有许多进行分裂的造孢组织细胞。第二年一月下旬至二月初小孢子母细胞进行减数分裂。在分裂期间,细胞内所贮存的淀粉粒的分布发生变化。二月初四分体小孢子形成,绒毡层细胞解体。二日中旬单核花粉粒形成,外壁扩展形成二个异极对称的气囊。三月花粉在四细胞时期散发。 雌配子体于二月上旬在珠心皮下分化出孢原细胞。二月下旬大孢子母细胞进入减数分裂期。三月初直列四分体大孢子形成,珠孔端三个退化,合点端一个功能大孢子进入有丝分裂期,形成约32个游离核的配子体。次年三月初雌配子体形成,四月初中央细胞核分裂,四月底颈卵器成熟,卵核周围产生辐射状原生质纤丝。五月初受精开始。云南松雌雄配子体的发育与亚热带分布的P.roburghii相似。     

Ovule and seed development of Lacandonia schismatica (Lacandoniaceae)

Ovule and seed development is described for Lacandonia schismatica, a species whose androecium is surrounded by the gynoecium. The ovule in each carpel is basal, anatropous, tenuinucellate, and bitegmic. The female gametophyte is formed by the micropylar megaspore cell, after a coenocytic stage of the four megaspore nuclei. The mature female gametophyte has the normal complement of seven cells and eight nuclei. We propose a new type of female gametophyte development on the basis of the coenocytic stage of the tetrad, the cellularization of the tetrad, and the survival of the micropylar spore. At seed dispersal time, the embryo has ~10-20 cells. Endosperm development is of the nuclear type. At maturity, endosperm cells show starch and protein inclusions as well as polysaccharides in their thick walls. The seed coat is formed from the outer integument; the inner one disappears. The exotesta contains tannin. The fruit (achene) wall is two-layered. The maturation of the fruits in a flower is synchronous, and they separate from the receptacle for dispersal.     

云贵水韭雌配子体发育及其濒危机制的研究

为了解云贵水韭(Isoetes yunguiensis)的生殖过程和濒危原因,采用人工培养获得其雌配子体,用半薄切片法观察雌配子体发育过程。结果表明,云贵水韭的大孢子萌发率约为26%;雌配子体异养,终生都在大孢子细胞壁内发育;颈卵器发育只有2枚颈沟细胞而无腹沟细胞。雌配子体发育停止在游离核时期、颈壁细胞数量偏少或排列紊乱、颈沟细胞异常、卵细胞凋亡等可能是导致生殖濒危的原因。同时,游离核是水韭生殖生物学研究和系统学研究的重要环节之一;缺失腹沟细胞可能是水韭类简化性进化的特征之一。     

Reproductive biology of henequen (Agave fourcroydes) and its wild ancestor Agave Angustifolia (Agavaceae). i. Gametophyte development

The pathways of micro- and megagametophyte development in Agave fourcroydes (henequén) and A. angustifolia were studied. We used histology and light microscopy to observe anther ontogeny and ovary differentiation in relation to flower bud size. Both species have the same sexual reproductive strategies and gametophyte development that may be divided into three phases: (1) premeiotic, which includes the establishment of the megaspore mother cell and the pollen mother cell; (2) meiotic, the formation of mature microspores and functional megaspores; (3) postmeiotic, which encompasses the development of mature pollen grains and the formation of the embryo sac. A successive type microsporogenesis was found in both species with formation of T-shaped tetrads and binuclear pollen grains. In vitro germination tests revealed very low pollen fertility. The female gametophyte is formed from two micropylar megaspore cells after the first meiotic division (bisporic type). Male and female gametogenesis occur asynchronously with microsporogenesis finishing before macrosporogenesis. The results so far show that the formation of male and female gametophytes in henequén is affected at different stages and that these alterations might be responsible for the low fertility shown by this species.     

Auxin Import and Local Auxin Biosynthesis Are Required for Mitotic Divisions,Cell Expansion and Cell Specification during Female Gametophyte Development in Arabidopsis thaliana

The female gametophyte of flowering plants, called the embryo sac, develops from a haploid cell named the functional megaspore, which is specified after meiosis by the diploid sporophyte. In Arabidopsis, the functional megaspore undergoes three syncitial mitotic divisions followed by cellularization to form seven cells of four cell types including two female gametes. The plant hormone auxin is important for sporophytic developmental processes, and auxin levels are known to be regulated by biosynthesis and transport. Here, we investigated the role of auxin biosynthetic genes and auxin influx carriers in embryo sac development. We find that genes from the YUCCA/TAA pathway (YUC1, YUC2, YUC8, TAA1, TAR2) are expressed asymmetrically in the developing ovule and embryo sac from the two-nuclear syncitial stage until cellularization. Mutants for YUC1 and YUC2 exhibited defects in cell specification, whereas mutations in YUC8, as well as mutations in TAA1 and TAR2, caused defects in nuclear proliferation, vacuole formation and anisotropic growth of the embryo sac. Additionally, expression of the auxin influx carriers AUX1 and LAX1 were observed at the micropylar pole of the embryo sac and in the adjacent cells of the ovule, and the aux1 lax1 lax2 triple mutant shows multiple gametophyte defects. These results indicate that both localized auxin biosynthesis and auxin import, are required for mitotic divisions, cell expansion and patterning during embryo sac development.     

大叶补血草的大、小孢子发生与雌、雄配子体的发育

系统地报道了大叶补血草(Limonium gmelinii (Willd.) Kuntze)的大、小孢子发生和雌、雄配子体的形成发育过程。主要结果如下：(1)小孢子母细胞减数分裂过程中的胞质分裂为同时型,四分孢子多为正四面体形, 也有少数为左右对称形;(2)成熟花粉为三细胞型,具3个萌发孔;(3)花药壁由5层细胞组成,最外层为表皮,其内分别为药室内壁、中层、绒毡层,绒毡层为变形型,花药壁的发育属于基本型;(4)大叶补血草的雌蕊由5心皮合生,子房1室,基生胎座,胚珠1个,拳卷型,双珠被,厚珠心;(5)孢原细胞发生于珠心表皮下,经一次平周分裂,形成造孢细胞,由造孢细胞直接发育成大孢子母细胞,大孢子母细胞减数分裂形成4个大孢子呈直线排列,合点端大孢子具功能,属于典型的蓼型胚囊发育。