EMBRYONIC FACTOR 19 encodes a pentatricopeptide repeat protein that is essential for the initiation of zygotic embryogenesis in Arabidopsis

Early embryogenesis is the most fundamental developmental process in biology.Screening of ethyl methanesulfonate(EMS)-mutagenized populations of Arabidopsis thaliana led to the identification of a zygote-lethal mutant embryonic factor 19(fac19)in which embryo development was arrested at the elongated zygote to octant stage.The number of endosperm nuclei decreased significantly in fac19 embryos.Genetic analysis showed fac19 was caused by a single recessive mutation with typical mendelian segregation,suggesting equal maternal and paternal contributions of FAC19 towards zygotic embryogenesis.Positional cloning showed that FAC19 encodes a putative mitochondrial protein with 16 conserved pentatricopeptide repeat(PPR)motifs.The fac19 mutation caused a conversion from hydrophilic serine located in a previously unknown domain to hydrophobic leucine.Crosses between FAC19/fac19 and the T-DNA insertion mutants in the same gene failed to complement the fac19 defects,confirming the identity of the gene.This study revealed the critical importance of a PPR protein-mediated mitochondrial function in early embryogenesis.     

Truncation of a Protein Disulfide Isomerase,PDIL2-1, Delays Embryo Sac Maturation and Disrupts Pollen Tube Guidance in Arabidopsis thaliana

Pollen tubes must navigate through different female tissues to deliver sperm to the embryo sac for fertilization. Protein disulfide isomerases play important roles in the maturation of secreted or plasma membrane proteins. Here, we show that certain T-DNA insertions in Arabidopsis thaliana PDIL2-1, a protein disulfide isomerase (PDI), have reduced seed set, due to delays in embryo sac maturation. Reciprocal crosses indicate that these mutations acted sporophytically, and aniline blue staining and scanning electron microscopy showed that funicular and micropylar pollen tube guidance were disrupted. A PDIL2-1-yellow fluorescent protein fusion was mainly localized in the endoplasmic reticulum and was expressed in all tissues examined. In ovules, expression in integument tissues was much higher in the micropylar region in later developmental stages, but there was no expression in embryo sacs. We show that reduced seed set occurred when another copy of full-length PDIL2-1 or when enzymatically active truncated versions were expressed, but not when an enzymatically inactive version was expressed, indicating that these T-DNA insertion lines are gain-of-function mutants. Our results suggest that these truncated versions of PDIL2-1 function in sporophytic tissues to affect ovule structure and impede embryo sac development, thereby disrupting pollen tube guidance.     

EMBRYONIC FACTOR 1 encodes an AMP deaminase and is essential for the zygote to embryo transition in Arabidopsis

Fusion of the egg and the sperm cells in plants produces a zygote that develops into an embryo. Screening of ethyl methanesulfonate-mutagenized populations of Arabidopsis led to the identification of EMBRYONIC FACTOR 1 (FAC1), a locus that gives a zygote-lethal phenotype when mutated. The FAC1 gene was identified by positional cloning and confirmed by a genetic complementation test against a T-DNA insertion allele. It encodes an AMP deaminase (AMPD) that is known in human and yeast to convert AMP to IMP to maintain the energy potential. Expression of FAC1 in a yeast AMPD mutant after removal of its N-terminal putative transmembrane domain complemented the mutant phenotype, suggesting a functional conservancy but a structural divergence through evolution. Although a low level of FAC1 expression was observed in all organs tested, using a reporter construct we observed a significantly increased FAC1 expression in the zygote, early embryo and endosperm. Furthermore, during somatic embryogenesis, a high level of FAC1 expression was observed in developing embryos including putative embryogenic cells. FAC1, therefore, represents one of the earliest expressed genes known in plants. It may act through AMP depletion to provide sufficient energy for the zygote to proceed through development.     

Arabidopsis hapless mutations define essential gametophytic functions

In flowering plants, the egg develops within a haploid embryo sac (female gametophyte) that is encased within the pistil. The haploid pollen grain (male gametophyte) extends a pollen tube that carries two sperm cells within its cytoplasm to the embryo sac. This feat requires rapid, precisely guided, and highly polarized growth through, between, and on the surface of the cells of the stigma, style, and ovary. Pollen tube migration depends on a series of long-range signals from diploid female cells as well as a short-range attractant emitted by the embryo sac that guides the final stage of tube growth. We developed a genetic screen in Arabidopsis thaliana that tags mutant pollen with a cell-autonomous marker carried on an insertion element. We found 32 haploid-disrupting (hapless) mutations that define genes required for pollen grain development, pollen tube growth in the stigma and style, or pollen tube growth and guidance in the ovary. We also identified genomic DNA flanking the insertion element for eleven hap mutants and showed that hap1 disrupts AtMago, a gene whose ortholog is important for Drosophila cell polarity.     

短柄五加开花后雌蕊的发育状态与受精作用的研究

短柄五加（ＥｌｅｕｔｈｅｒｏｃｏｃｕｓｂｒａｃｈｙｐｕｓＨａｒｍｓ．）开花当天,花药散粉,而雌配子体需经４～５ｄ才发育成熟。证实短柄五加为雄蕊先熟植物。开花第５天,成熟胚囊的比率为５７６９％,其余为退化和不育胚囊。开花第６天,胚囊开始受精。开花第１０天,受精胚囊占胚囊总数的５３５７％。柱头的可授期自开花后第４～５天开始,自花粉萌发至雌雄性核融合大约有２～３ｄ的间隔期。短柄五加受精过程与一般被子植物相同,其受精作用属于有丝分裂前配子融合类型。观察并统计了合子中雌性核仁的数目、存在状态,指出短柄五加合子中从雄性核仁出现到与雌雄性核仁融合为一个大核仁需经历３ｄ左右;如果以胚乳游离核数目为对照,大部分合子中雌雄性核仁的融合发生在３２～１２８个胚乳游离核时期。大多数合子是以雌雄性核仁融合为一个大核仁后进入合子分裂期;少数合子的雌雄性核仁不经融合也进入合子分裂期。观察到多精入胚囊、多精入卵以及成熟胚囊退化的现象。讨论了被子植物受精过程中有关受精终结的标志等问题。     

Embryo and endosperm development is disrupted in the female gametophytic capulet mutants of Arabidopsis

The female gametophyte of higher plants gives rise, by double fertilization, to the diploid embryo and triploid endosperm, which develop in concert to produce the mature seed. What roles gametophytic maternal factors play in this process is not clear. The female-gametophytic effects on embryo and endosperm development in the Arabidopsis mea, fis, and fie mutants appear to be due to gametic imprinting that can be suppressed by METHYL TRANSFERASE1 antisense (MET1 a/s) transgene expression or by mutation of the DECREASE IN DNA METHYLATION1 (DDM1) gene. Here we describe two novel gametophytic maternal-effect mutants, capulet1 (cap1) and capulet2 (cap2). In the cap1 mutant, both embryo and endosperm development are arrested at early stages. In the cap2 mutant, endosperm development is blocked at very early stages, whereas embryos can develop to the early heart stage. The cap mutant phenotypes were not rescued by wild-type pollen nor by pollen from tetraploid plants. Furthermore, removal of silencing barriers from the paternal genome by MET1 a/s transgene expression or by the ddm1 mutation also failed to restore seed development in the cap mutants. Neither cap1 nor cap2 displayed autonomous seed development, in contrast to mea, fis, and fie mutants. In addition, cap2 was epistatic to fis1 in both autonomous endosperm and sexual development. Finally, both cap1 and cap2 mutant endosperms, like wild-type endosperms, expressed the paternally inactive endosperm-specific FIS2 promoter GUS fusion transgene only when the transgene was introduced via the embryo sac, indicating that imprinting was not affected. Our results suggest that the CAP genes represent novel maternal functions supplied by the female gametophyte that are required for embryo and endosperm development.     

The Development of Gynoecium After Anthesis and Fertilization in Eleutherococcus brachypus (Araliaceae)

Eleutherococcus brachypus Harms. is a protandrous plant because the female gametophyte delays its maturation until the fifth day after anthesis and pollen shelling. On the fifth day after anthesis, about 57.69% of the embryo sacs matured and the rest degenerated or failed to develop. Fertilization began in the embryo sac on the fifth day. On the tenth day fertilization took place in 53.37 % of the total of embryo sacs. The stigma became receptible after 3 to 4 days of anthesis. It took 2 to 3 days from the germination of pollen grains on stigma to the fusion of male and female nuclei. The process of fertilization in E. brachypus is not different from most other angiosperms. It belonged to the type of premitotic syngamy. The observations and statistical analysis were made on the number feature of male and female nucleoli in the zygote. The result indicated that it took three days or so from the appearance of male nucleolus in the zygote to its fusion with the female nucleotus. Refering to the number of free nuclei of the endosperm, the fusion of male and female nucleoli in most of the zygotes occurred in the stage of 32 to 128 nuclei of the endosperm. Most zygotes con-tained a big nucleolus resulting from the fusion of male and female nucleolus and proceeding to mitosis. A few without fusion could also proceed to the mitotic stage. Features of multiple sperms entering the embryo sac or entering the egg cell and the degeneration of mature embryo sacs were observed as well. The sign of the termination of fertilization in angiosperms was discussed.     

Isolation of GUS marker lines for genes expressed in Arabidopsis endosperm,embryo and maternal tissues

In order to identify marker lines expressing GUS in various endosperm compartments and at different developmental stages, a collection of Arabidopsis thaliana (L.) Heynh. promoter trap lines were screened. The screen identified 16 lines displaying GUS-reporter gene expression in the endosperm, embryo and other seed organs. The distinctive patterns of GUS expression in these lines provide molecular markers for most cell compartments in the endosperm of Arabidopsis seeds at all developmental stages, and represent a valuable research tool for characterizing present and future Arabidopsis seed mutants. GUS expression patterns of these 16 lines are presented here. One line showed chalazal endosperm-specific GUS activity at the heart stage of embryo development. In six lines embryo-specific GUS activity was detected. Six lines exhibited GUS activity predominantly in the endosperm and embryo while two lines showed strong GUS activity in all seed organs. In one line GUS activity was detected in integuments and syncytial endosperm, while the GUS activity at the cotyledonary stage of the embryo was seed coat-specific. In addition, two funiculus markers and two silique markers expressed in the abscission zone and the guard cells are also presented.     

Arabidopsis GLAUCE promotes fertilization-independent endosperm development and expression of paternally inherited alleles

Early seed development of sexually reproducing plants requires both maternal and paternal genomes but is prominently maternally influenced. A novel gametophytic maternal-effect mutant defective in early embryo and endosperm development, glauce (glc), has been isolated from a population of Arabidopsis Ds transposon insertion lines. The glc mutation results from a deletion at the Ds insertion site, and the molecular identity of GLC is not known. glc embryos can develop up to the globular stage in the absence of endosperm and glc central cells appear to be unfertilized. glc suppresses autonomous endosperm development observed in the fertilization-independent seed (fis) class mutants. glc is also epistatic to mea, one of the fis class mutants, in fertilized seeds, and is essential for the biparental embryonic expression of PHE1, a repressed downstream target of MEA. In addition, maternal GLC function is required for the paternal embryonic expression of the ribosome protein gene RPS5a and the AMP deaminase gene FAC1, both of which are essential for early embryo and endosperm development. These results indicate that factors derived from the female gametophyte activate a subset of the paternal genome of fertilized seeds.     

The plant-specific ssDNA binding protein OSB1 is involved in the stoichiometric transmission of mitochondrial DNA in Arabidopsis

Plant mitochondrial genomes exist in a natural state of heteroplasmy, in which substoichiometric levels of alternative mitochondrial DNA (mtDNA) molecules coexist with the main genome. These subgenomes either replicate autonomously or are created by infrequent recombination events. We found that Arabidopsis thaliana OSB1 (for Organellar Single-stranded DNA Binding protein1) is required for correct stoichiometric mtDNA transmission. OSB1 is part of a family of plant-specific DNA binding proteins that are characterized by a novel motif that is required for single-stranded DNA binding. The OSB1 protein is targeted to mitochondria, and promoter-beta-glucuronidase fusion showed that the gene is expressed in budding lateral roots, mature pollen, and the embryo sac of unfertilized ovules. OSB1 T-DNA insertion mutants accumulate mtDNA homologous recombination products and develop phenotypes of leaf variegation and distortion. The mtDNA rearrangements occur in two steps: first, homozygous mutants accumulate subgenomic levels of homologous recombination products; second, in subsequent generations, one of the recombination products becomes predominant. After the second step, the process is no longer reversible by backcrossing. Thus, OSB1 participates in controlling the stoichiometry of alternative mtDNA forms generated by recombination. This regulation could take place in gametophytic tissues to ensure the transmission of a functional mitochondrial genome.     

罗汉果双受精过程的细胞学观察

罗汉果（Ｓｉｒａｉｔｉａｇｒｏｓｖｅｎｏｒｉ（Ｓｗｉｎｇｌｅ）Ｃ．Ｊｅｍｅｙ）双受精过程属有丝分裂前配子融合类型,授粉后２４～４８ｈ,花粉管进入胚囊,穿过一个助细胞,放出两个精子。雌雄核融合和雄核与次生核融合同时发生在授粉后６２～７２,雄核与次生核融合速度快于配子融合,７２ｈ后即可见到初生胚乳核分裂。合子中的雌雄核仁在授粉后第５～６ｄ融合,授粉后８～９ｄ合成分裂形成二细胞胚。在双受精过程中,多次观察到有多条花粉管进入胚囊和多精入极核现象。原胚期有附加花粉管从珠孔进入。     

The MADS domain protein DIANA acts together with AGAMOUS-LIKE80 to specify the central cell in Arabidopsis ovules

MADS box genes in plants consist of MIKC-type and type I genes. While MIKC-type genes have been studied extensively, the functions of type I genes are still poorly understood. Evidence suggests that type I MADS box genes are involved in embryo sac and seed development. We investigated two independent T-DNA insertion alleles of the Arabidopsis thaliana type I MADS box gene AGAMOUS-LIKE61 (AGL61) and showed that in agl61 mutant ovules, the polar nuclei do not fuse and central cell morphology is aberrant. Furthermore, the central cell begins to degenerate before fertilization takes place. Although pollen tubes are attracted and perceived by the mutant ovules, neither endosperm development nor zygote formation occurs. AGL61 is expressed in the central cell during the final stages of embryo sac development. An AGL61:green fluorescent protein–β-glucoronidase fusion protein localizes exclusively to the polar nuclei and the secondary nucleus of the central cell. Yeast two-hybrid analysis showed that AGL61 can form a heterodimer with AGL80 and that the nuclear localization of AGL61 is lost in the agl80 mutant. Thus, AGL61 and AGL80 appear to function together to differentiate the central cell in Arabidopsis. We renamed AGL61 DIANA, after the virginal Roman goddess of the hunt.     

The alpha-N-acetyl-glucosaminidase gene is transcriptionally activated in male and female gametes prior to fertilization and is essential for seed development in Arabidopsis

Sugar residues in proteoglycan complexes carry important signalling and regulatory functions in biology. In humans, heparan sulphate is an example of such a complex polymer containing glucosamine and N-acetyl-glucosamine residues and is present in the extracellular matrix. Although heparan sulphate has not been found in plants, the At5g13690 gene encoding the alpha-N-acetyl-glucosaminidase (NAGLU), an enzyme involved in its catabolism, is present in the Arabidopsis genome. Among our collection of embryo-defective lines, a plant was identified in which the T-DNA had inserted into the AtNAGLU gene. The phenotype of atnaglu is an early arrest of seed development without apparent male or female gametophytic effects. These data demonstrated the essential function in Arabidopsis consistent with the contribution of NAGLU to the Sanfilippo syndrome in human. Expression of AtNAGLU in plants was shown to be prevalent during reproductive development. The presence of AtNAGLU mRNA was observed during early and late male gametogenesis and in each cell of the embryo sac at the time of fertilization. After fertilization, AtNAGLU was expressed in the embryo, suspensor, and endosperm until the cotyledonary stage embryo. This precise pattern of expression identifies the cells and tissues where a remodelling of the N-acetyl-glucosamine residues of proteoglycan complexes is occurring. This work provides original evidence of the important role of N-acetyl-glucosamines in plant reproductive development.     

Fertilization and Histochemical Investigation on Pulsatilla chinensis (Bung) Regel

Fertilization and variation of protein and starch grains in Pulsatilla chinensis (Bung) Regel have been studied at light microscopic level with histochemical test. Based upon the observations, the main conclusions are summarized as follows: The mature pollen grains are two-celled in which the generative cell shows the stronger protein staining than the vegetative cell. And vegetative cells are full of starch garins. When the pollen tube enters into the embryo sac, one synergid is destroyed, or in a few cases synergids are intact. Occasionally two synergids are disorganized as pollen tube penetrates. However, most of the remaining syuergids break down during fertilization, only in a few cases it remains till early stage of embryo development. The contents discharged by the pollen tube consist of two sperms, which stain intensely blue with protein dyes, a great amount of protein and starch grains. Mature female gametophyte (embryo sac) consists of an egg apparatus, central cell, which has a huge secondary nucleus, and antipodal apparatus which retain in course of fertilization. A few of embryo sac contain two sets of egg apparatus, a central cell with two huge secondary nuclei and two sets of antipodal apparatus. In some nucleoli of the central cell the comb-like structure pattern may be detected clearly. There are 1–2 small nucleoli in some egg cells and central cells. All the cells in embryo sac show protein positive reaction. According to the different shades of the color in cells, its may be arranged in the following order: antipodal cells, synergids, central cell and egg cell. Only a few small starch grains are present near nuclei of central cell and egg cell before fertilization, but no starch grains remain in most of the central cell, the synergids and antipodal cells. The fertilization is of the premitotic type. The fusion of the sexual nuclei progresses in the following order: 1, sperms approach and lie on the egg nucleus and secondary nucleus; 2, sperm chromatin sinks themselves into female nucleus, and male nucleolus emerges with the sperm chromosome; and 3, male nucleoli fuse with the nucleoli of egg nucleus and central cell nucleus, and finally forming the zygote and the primary endosperm cells respectively. Nevertheless, as it is well known, the fertilization completes in central cell obviously earlier than that in egg cell. Though it has been explained in cereals and cotton, in Pulsatilla chinensis the main reason is that nucleolar fusion of the male and female nucleoli in egg nucleus is slower than that in secondary nucleus. And the dormancy of the primary endosperm nucleus is shorter than that of the zygote. In the process of fertilization, histochemical changes are considerably obvious in the following three parts: 1, from the begining of fusion of male and female nuclei to form zygote and primary endosperm cell, Protein staining around female nucleus appears to increase gradually; 2, no starch grains are detected in embryo sac. Though only starch grains are carried in by pollen tube, they are completely exhausted during this period; and 3, near completion of fertilization starch grains appear again in zygote, however, not yet in primary endosperm nucleus till its dividing for the first time. The present study reveals that antipodal cells and synergids seem to play a significant role in nutrition of the embryo sac during the fertilization.