EMBRYONIC FACTOR 31 encodes a tyrosyl-tRNA synthetase that is essential for seed development

Aminoacyl-tRNA synthetases (AARSs) involve the process of catalyzing the ligation of specific amino acids to their cognate tRNAs. Here we identified an Arabidopsis mutant embryonic factor 31 (fac31), its embryos arrested at development from one cell to globular stage. The FAC31 gene was identified by positional cloning and confirmed by a genetic complementation test with two independent T-DNA insertion lines and transgenic rescue with full-length genomic DNA. FAC31 encodes a Tyrosyl-tRNA synthetase and localize to mitochondria and cytoplasm. Fac31 mutants contain a point mutation from CAA to a stop codon TAA which may lead to a truncated protein. The phenotype of fac31 mutants are very similar to the T-DNA insertion lines Salk_016722 and Salk_045570 displayed smaller embryo sac contains only less number of endosperm nucleolus. Genetic analysis showed that the FAC31 gene had no parental effects through the transmission of mutated FAC31 gene by gametes. FAC31 is a high-conserved protein among animals and plants. RT-PCR analysis and promoter-GUS expression showed that it is expressed in nearly all tissues tested, strongly expressed in meristem of seedlings, the primordium of lateral root, young inflorescences, mature pollen, germinated pollen tubes and embryo sacs before heart stage. Our findings suggest that FAC31 is essential for the seed development through regulation the expanding of embryo sac and proliferation of endosperm nucleolus.     

Immunolocalization of arabinogalactan proteins inAmaranthus hypochondriacus L. ovules

Summary Amaranthus hypochondriacus ovules are of the crassinucellate type, having several layers of nucellus cells between the micropyle and the embryo sac through which pollen tubes have to penetrate. The ultrastructural features of the micropylar nucellus cells appear to reflect cells with high metabolic activity. With the monoclonal antibodies MAC207 and JIM8 (against arabinogalactan proteins) we have shown that the presence of the two epitopes was different in the gametophytic tissues and embryo sac. The young embryo and suspensor cells are reactive only to Mab JIM8. The selective presence and localization of these two epitopes was also demonstrated in the micropylar nucellus cells. The expression of these arabinogalactan proteins in this ovule seems to be closely aligned with the pathway of the pollen tube, possibly providing directional guides for tube growth inside the ovule.     

Protein Disulfide Isomerase-Like Protein 1-1 Controls Endosperm Development through Regulation of the Amount and Composition of Seed Proteins in Rice

Protein disulfide isomerase (PDI) is a chaperone protein involved in oxidative protein folding by acting as a catalyst and assisting folding in the endoplasmic reticulum (ER). A genome database search showed that rice contains 19 PDI-like genes. However, their functions are not clearly identified. This paper shows possible functions of rice PDI-like protein 1-1 (PDIL1-1) during seed development. Seeds of the T-DNA insertion PDIL1-1 mutant, PDIL1-1Δ, identified by genomic DNA PCR and western blot analysis, display a chalky phenotype and a thick aleurone layer. Protein content per seed was significantly lower and free sugar content higher in PDIL1-1Δ mutant seeds than in the wild type. Proteomic analysis of PDIL1-1Δ mutant seeds showed that PDIL1-1 is post-translationally regulated, and its loss causes accumulation of many types of seed proteins including glucose/starch metabolism- and ROS (reactive oxygen species) scavenging-related proteins. In addition, PDIL1-1 strongly interacts with the cysteine protease OsCP1. Our data indicate that the opaque phenotype of PDIL1-1Δ mutant seeds results from production of irregular starch granules and protein body through loss of regulatory activity for various proteins involved in the synthesis of seed components.     

Mutation in SUMO E3 ligase, SIZ1, disrupts the mature female gametophyte in Arabidopsis

Female gametophyte is the multicellular haploid structure that can produce embryo and endosperm after fertilization, which has become an attractive model system for investigating molecular mechanisms in nuclei migration, cell specification, cell-to-cell communication and many other processes. Previous reports found that the small ubiquitin-like modifier (SUMO) E3 ligase, SIZ1, participated in many processes depending on particular target substrates and suppression of salicylic acid (SA) accumulation. Here, we report that SIZ1 mediates the reproductive process. SIZ1 showed enhanced expression in female organs, but was not detected in the anther or pollen. A defect in the siz1-2 maternal source resulted in reduced seed-set regardless of high SA concentration within the plant. Moreover, aniline blue staining and scanning electron microscopy revealed that funicular and micropylar pollen tube guidance was arrested in siz1-2 plants. Some of the embryo sacs of ovules in siz1-2 were also disrupted quickly after stage FG7. There was no significant affects of the siz1-2 mutation on expression of genes involved in female gametophyte development- or pollen tube guidance in ovaries. Together, our results suggest that SIZ1 sustains the stability and normal function of the mature female gametophyte which is necessary for pollen tube guidance.     

MICROPYLAR EXUDATE IN GASTERIA (ALOACEAE) AND ITS POSSIBLE FUNCTION IN POLLEN TUBE GROWTH

The micropylar exudate of Gasteria verrucosa (Mill.) H. Duval was studied using light and electron microscopic techniques. Ovules may contain micropylar exudate before stigma receptivity. During successive phases of stigma receptivity, the number of ovules with micropylar exudate and the amount of micropylar exudate per ovule increases. At the late phase of stigma receptivity, large amounts of micropylar exudate with a smooth to cauliflowerlike appearance were observed. Micropylar exudate is viscous and contains, among other components, proteins and carbohydrates. At all stages of the stigma investigated, ovules situated at the base of the ovary contain a larger quantity of micropylar exudate than those at the top. The appearance of micropylar exudate is related to the degree of development of the embryo sac and it originates primarily from the filiform apparatus. It is assumed that an uptake of water by the ovule initiates the outflow of micropylar exudate from the filiform apparatus into the micropyle. Both ovular pollen tube ingrowth and seed set mark the optimum pollination stage of the stigma, which for both events lies around the onset of stigma receptivity. When pollen tubes have reached the ovary, young micropylar exudate stimulates their growth rate. The presence of micropylar exudate seems to be a requirement for pollen tube penetration, and an interaction between the pollen tube and the micropylar exudate has been proposed. Possibly, the micropylar exudate serves as a nutritional source and, in an optimum condition, as an attractant for approaching pollen tubes.     

GABA,a new player in the plant mating game

Pollen tubes are guided through female tissues to deliver sperms to the embryo sac. A recent study reveals a GABA gradient along the pollen tube path, which, together with guidance defects in GABA-overaccumulating mutants, implies a role for GABA in regulating pollen tube growth and guidance.     

花粉管引导机制的研究进展

花粉管引导是指显花植物在受精过程中,雌蕊组织与花粉管相互作用使花粉管定向生长并最终到达胚囊的过程,其机制颇为复杂。该文基于调控花粉管生长的孢子体引导和配子体细胞引导两个主要过程,阐述雌蕊中不同蛋白分子和其它小分子物质的浓度梯度在花粉管的孢子体组织引导中的作用,以及胚囊中不同类型的细胞及其相关基因与蛋白在花粉管的配子体细胞引导中的作用。同时,该文也对精细胞在花粉管引导中的作用进行了阐述。     

External application of gametophyte-specific ZmPMEI1 induces pollen tube burst in maize

Regulated demethylesterification of homogalacturonan, a major component of plant cell walls, by the activity of pectin methylesterases (PMEs), plays a critical role for cell wall stability and integrity. Especially fast growing plant cells such as pollen tubes secrete large amounts of PMEs toward their apoplasmic space. PME activity itself is tightly regulated by its inhibitor named as PME inhibitor and is thought to be required especially at the very pollen tube tip. We report here the identification and functional characterization of PMEI1 from maize (ZmPMEI1). We could show that the protein acts as an inhibitor of PME but not of invertases and found that its gene is strongly expressed in both gametophytes (pollen grain and embryo sac). Promoter reporter studies showed gene activity also during pollen tube growth toward and inside the transmitting tract. All embryo sac cells except the central cell displayed strong expression. Weaker signals were visible at sporophytic cells of the micropylar region. ZmPMEI1–EGFP fusion protein is transported within granules inside the tube and accumulates at the pollen tube tip as well as at sites where pollen tubes bend and/or change growth directions. The female gametophyte putatively influences pollen tube growth behavior by exposing it to ZmPMEI1. We therefore simulated this effect by applying recombinant protein at different concentrations on growing pollen tubes. ZmPMEI1 did not arrest growth, but destabilized the cell wall inducing burst. Compared with female gametophyte secreted defensin-like ZmES4, which induces burst at the very pollen tube tip, ZmPMEI1-induced burst occurs at the subapical region. These findings indicate that ZmPMEI1 secreted by the embryo sac likely destabilizes the pollen tube wall during perception and together with other proteins such as ZmES4 leads to burst and thus sperm release.     

丹东蒲公英专性无融合生殖特性

采用田间去雄套袋、花粉活性与花粉管萌发观测等交配系统实验,辅以染色体分析、胚囊发育观察以及流式细胞仪检测,对丹东蒲公英(Taraxacum antungense)生殖特性进行系统研究。结果表明,去雄套袋情况下,结籽率高达96%,由此证明交配系统具有极高的无融合发生率;自花授粉不能萌发,花粉活性仅为20.6%,异花授粉虽可萌发,但生长缓慢,花粉在到达子房前,花粉管已停止生长并消失;二分体时期,合子端大孢子发育为功能性大孢子,珠孔端大孢子退化消失,经蓼型发育,形成7细胞8核,卵细胞继续发育形成原胚,中央细胞形成胚乳,其它细胞退化消失,花未开放前,已形成球形胚。此外,丹东蒲公英为三倍体。流式细胞仪检测结果表明,丹东蒲公英为专性无融合生殖,胚乳自主发育。综合以上研究结果,说明丹东蒲公英具专性无融合生殖特性。     

东北地区野豌豆属VICIAL．物种生物学研究Ⅷ．幼苗形体结构动态和种间界限

在野外居群调查的启示下,本文以组件观点对柳叶野豌豆复合种和歪头菜幼苗亚单位的时序变化与开花关系进行了分析。结果发现在柳叶野豌豆复合种栽培居群中存在打破物种间形体结构特征的个体,即在复叶由一对小叶组成的植株就已开花而进入生殖时期。另外,在歪头菜的野生居群中发现由三或四枚小叶组成复叶的个体,因此,我们推测这种形体结构的变化可能暗示着柳叶野豌豆复合种和歪头菜有着共同的祖先。     

Structural characteristics of the mature embryo sac of Camellia oleifera

The ovule is the most important reproductive organ in the pistil of phanerogamae. Camellia oleifera (Theaceae) is an important woody plant producing edible oil in southern China, and its embryo sac structure has a positive effect on seed breeding. In this study, the microstructure, ultrastructure and three‐dimensional structure of the ovule and embryo sac of C. oleifera were observed and described based on a combination of advanced microscopy techniques (SEM, TEM, CLSM). The ovule comprises the inner and outer integument. Large quantities of secretions in the micropylar canal exit and may participate in the guidance of the entry of the pollen tube into the embryo sac. The synergids have a dense cytoplasm, abundant organelles, and strong polarity. Little cytoplasm is present in the egg cell, yet there are many vacuoles. The center of the cell is taken up by a large vacuole, and the cytoplasm is pushed towards the edges to form obvious cytoplasmic cords. The two polar nuclei are large and conspicuous. The antipodal cells degenerate to fulfill a nutritional function.     

Ultrastructure of the Developing and Fertilized Embryo Sac of Amaranthus hypochondriacus L.

Amaranthus hypochondriacus embryo sac development was investigatedbefore and after fertilization. During the early stages of development,the young embryo sac displays three antipodal cells at the chalazalpole that degenerate very early in the maturation process, beforethe synergids and egg cell are completely differentiated. Themature embryo sac is composed only of the female germ unit.The synergid cells organize a filiform apparatus accompaniedby the presence of mitochondria and dictyosomes with numerousvesicles. The involvement of the synergids in transport andsecretory functions related to pollen tube attraction and guidance,are discussed. The egg cell is located at the micropylar polenear the synergids and displays exposed plasma membranes atthe chalazal pole. The fertilized egg cell does not exhibitmarked changes after fertilization except for the closure ofthe cell wall. The central cell is the largest cell of thisvery long embryo sac. The fused nucleus is close to the eggapparatus before fertilization and displays a remarkable chalazalmigration after gamete delivery. The ultrastructure of the centralcell cytoplasm and the numerous wall ingrowths around this cellsuggest an important role in nutrient transportation. Aftergamete delivery, the embryo sac displays electron dense bodiesthat aggregate within the intercellular space between the synergids,egg cell and central cell. These bodies, that appear in theembryo sac of several plants, are probably involved in gametedelivery for double fertilization. The possibility of biparentalinheritance of mitochondria in this plant is also discussed.Copyright 1999 Annals of Botany Company Amaranthus hypochondriacus, grain amaranth, embryo sac, fertilization.     

Guidance in vitro of the pollen tube to the naked embryo sac of torenia fournieri

The precise guidance of the pollen tube to the embryo sac is critical to the successful sexual reproduction of flowering plants. We demonstrate here the guidance of the pollen tube to the embryo sac in vitro by using the naked embryo sac of Torenia fournieri, which protrudes from the micropyle of the ovule. We developed a medium for culture of both the ovule and the pollen tube of T. fournieri and cocultivated them in a thin layer of solid medium. Although pollen tubes that had germinated in vitro passed naked embryo sacs, some pollen tubes that grew semi-in vitro through a cut style arrived precisely at the site of entry into the embryo sac, namely, the filiform apparatus of the synergids. When pollen tubes were unable to enter the embryo sac, they continuously grew toward the same filiform apparatus, forming narrow coils. Pollen tubes selectively arrived at complete, unfertilized embryo sacs but did not arrive at those of heat-treated ovules or those with disrupted synergids. These results convincingly demonstrate that pollen tubes are specifically attracted to the region of the filiform apparatus of living synergids in vitro.     

Distinct roles of protein disulfide isomerase and P5 sulfhydryl oxidoreductases in multiple pathways for oxidation of structurally diverse storage proteins in rice

In the rice (Oryza sativa) endosperm, storage proteins are synthesized on the rough endoplasmic reticulum (ER), in which prolamins are sorted to protein bodies (PBs) called type-I PB (PB-I). Protein disulfide isomerase (PDI) family oxidoreductase PDIL2;3, an ortholog of human P5, contains a conserved structural disulfide in the redox-inactive thioredoxin-like (TRX) domain and was efficiently targeted to the surface of PB-I in a redox active site-dependent manner, whereas PDIL1;1, an ortholog of human PDI, was localized in the ER lumen. Complementation analyses using PDIL1;1 knockout esp2 mutant indicated that the a and a' TRX domains of PDIL1;1 exhibited similar redox activities and that PDIL2;3 was unable to perform the PDIL1;1 functions. PDIL2;3 knockdown inhibited the accumulation of Cys-rich 10-kD prolamin (crP10) in the core of PB-I. Conversely, crP10 knockdown dispersed PDIL2;3 into the ER lumen. Glutathione S-transferase-PDIL2;3 formed a stable tetramer when it was expressed in Escherichia coli, and the recombinant PDIL2;3 tetramer facilitated α-globulin(C79F) mutant protein to form nonnative intermolecular disulfide bonds in vitro. These results indicate that PDIL2;3 and PDIL1;1 are not functionally redundant in sulfhydryl oxidations of structurally diverse storage proteins and play distinct roles in PB development. We discuss PDIL2;3-dependent and PDIL2;3-independent oxidation pathways that sustain disulfide bonds of crP10 in PB-I.     

Changes of calcium distribution in ovules ofTorenia fournieri during pollination and fertilization

Summary Calcium distribution in ovules ofTorenia fournieri was studied by electron energy loss spectroscopy and transmission electron microscopic visualization of calcium antimonate precipitates. High calcium levels were found in the ovules ofT. fournieri. Calcium is situated mainly in extracellular regions before fertilization, including the surface of embryo sac, in the mucilage, and among the cells of the egg apparatus. Intracellular calcium was found only in the nucellar cells around the embryo sac and in the epidermis of the central axis and funiculus. After pollination, a labyrinthine structure (coralloid-like cell wall formation) develops on the micropylar surfaces of the egg apparatus that contain high levels of calcium. Calcium levels increase in the degenerating synergid after the penetration of the pollen tube. Calcium-antimonate precipitates are abundant in vacuoles of the disrupted synergid and pollen tube cytoplasm.Abbreviations EELS electron energy loss spectroscopy - EDX energy-dispersive X-ray microanalysis - LS labyrinthine structure     

Ovary signals for directional pollen tube growth

In angiosperms, the female gametophyte has a secluded life; it is protected by several concentric layers that envelop each other. The embryo sac is surrounded by the nucellus, which in turn is wrapped by the integuments forming the ovule, which is nested in the ovary. These wrappings are not hermetic, but contain little ”gates” the pollen tube must traverse on its way towards the embryo sac. Information is emerging that shows that the ovary and ovule provide signals orienting and directimng the pollen tube on the right course. There are three main bodies of evidence supporting this hypothesis. One relates to developmental changes in the female tissues and how they affect pollen tube growth. The second refers to defective ovule mutants, which induce defective pollen tube guidance. And the third relates to the possible molecules involved in this signalling. Here, information gathered along these three main lines of evidence is reviewed. All converge to the conclusion that different checkpoints exist all along the pollen tube pathway. These checkpoints provide active signalling that guides the pollen tube to its destination, the embryo sac. Received: 15 December 2000 / Accepted: 13 June 2001     

Polar distribution dynamics of Con A binding sites in embryo sacs is temporally coupled by the fertilization process

The binding site distribution of concanavalin agglutinin (Con A) and wheat germ agglutinin (WGA) on embryo sacs at various developmental stages of Torenia fournieri L was studied by using a cooled Charge Coupled Device (CCD) and fluorescent Con A and WGA probes. The distribution patterns of Con A and WGA binding sites on embryo sacs changed during the fertilization process. The fluorescent signal indicating Con A binding sites was distributed evenly on the surface of the embryo sac wall before anthesis, was much denser on the micropylar end of the embryo sac wall and looked like a corona on the day of anthesis. After pollination, stronger fluorescence was present on the micropylar end of the embryo sac wall and the filiform apparatus (FA), showing an obvious polar distribution. When the pollen tube entered the embryo sac and reached a synergid, the fluorescence was still concentrated on the micropylar end and FA, and started to appear on the synergid. After fertilization, the polar distribution of the fluorescence gradually disappeared and an even distribution pattern was observed again on the embryo sac wall. These results revealed that the dynamic distribution of Con A binding sites was temporally coupled with the process of fertilization. WGA binding site distribution on the embryo sac was also investigated and showed a simple pattern but also regularly changed during the process of fertilization. The variation of these lectin binding sites during the fertilization process suggests that lectin binding site interactions may play a role in the process.