Advances in the study of zygote activation in higher plants.

In higher plants, fertilization induces many structural and physiological changes in the fertilized egg that reflect the transition from the haploid female gamete to the diploid zygote - the first cell of the sporophyte. After fusion of the egg nucleus with the sperm nucleus, many molecular changes occur in the zygote during the process of zygote activation during embryogenesis. The zygote originates from the egg, from which some pre-stored translation initiation factors transfer into the zygote and function during zygote activation. This indicates that the control of zygote activation is pre-set in the egg. After the egg and sperm nuclei fuse, gene expression is activated in the zygote, and paternal and maternal gene expression patterns are displayed. This highlights the diversity of zygotic genome activation in higher plants. In addition to new gene expression in the zygote, some genes show quantitative changes in expression. The asymmetrical division of the zygote produces an apical cell and a basal cell that have different destinies during plant reconstruction; these destinies are determined in the zygote. This review describes significant advances in research on the mechanisms controlling zygote activation in higher plants.

Isolation of male and female gametes, zygotes and proembryos of leek (Allium tuberosum Roxb).

The isolation of male and female gametes is an effective method to study the fertilization mechanisms of higher plants. An osmotic shock method was used to rupture pollen grains of Allium tuberosum Roxb and release the pollen contents, including generative cells, which were mass collected. The pollinated styles were cut following 3 h of in vivo growth, and cultured in medium for 6-8 h, during which time pollen tubes grew out of the cut end of the style. After pollen tubes were transferred into a solution containing 6% mannitol, tubes burst and released pairs of sperm cells. Ovules of A. tuberosum were incubated in an enzyme solution for 30 min, and then dissected to remove the integuments. Following transfer to a dissecting solution free of enzymes, each nucellus was cut in the middle, and squeezed gently on the micropylar end, resulting in the liberation of the egg, zygote and proembryo from ovules at selected stages. These cells can be used to explore fertilization and embryonic development using molecular biological methods for each cell type and development stage.

Wheat egg in vitro fusion with wheat and green bristlegrass sperm.

SummaryIsolated gametes can be used to investigate fertilization mechanisms, and probe distant hybridization between different species. Pollen grains of wheat and Setaria viridis are tricellular, containing sperm cells at anthesis. Sperm from these plants were isolated by breaking open pollen grains in a osmotic solution. Wheat ovules were digested in an enzyme solution for 20 min, and then transferred to an isolation solution without enzymes to separate egg cells from ovules. The fusion of wheat egg cells with wheat and S. viridis sperm was conducted using an electro-fusion apparatus. Under suitable osmotic pressure (10% mannitol), calcium concentration of 0.001% (CaCl2·2H2O), and a 30-35 V alternating electric field for 15 s, egg cells and sperm adhered to each other and became arranged in a line. Electroporation of the plasma membrane of egg cells and sperm using a 300-500 V direct-current electric field (45 µs amplitude pulse) caused them to fuse.

Calcium: A Critical Factor in Pollen Germination and Tube Elongation.

Pollen is the male gametophyte of higher plants. Its major function is to deliver sperm cells to the ovule to ensure successful fertilization. During this process, many interactions occur among pollen tubes and pistil cells and tissues, and calcium ion (Ca2+) dynamics mediate these interactions among cells to ensure that pollen reaches the embryo sac. Although the precise functions of Ca2+ dynamics in the cells are unknown, we can speculate about its roles on the basis of its spatial and temporal characteristics during these interactions. The results of many studies indicate that calcium is a critical element that is strongly related to pollen germination and pollen tube growth.

Advances in the study of egg activation of higher plants.

Fertilization in higher plants induces many structural and physiological changes in the fertilized egg, and represents the transition from the haploid female gamete to the diploid zygote, the first cell of a sporophyte. Some changes are induced extremely rapidly following fusion with sperm cells and are the preclusions of egg activation. This review focuses on the early changes that occur in the egg after fusion with sperm cells, but before nuclear fusion. Reported changes include cell shrinkage, cell wall formation, polarity change, oscillation in Ca2+ concentration, and DNA synthesis. In addition, the current understanding of egg activation is summarized and the possible functional relevance of the changes is explored.

Calcium controls the formation of vacuoles from mitochondria to regulate microspore development in wheat.

Potassium antimonite was used to investigate the localisation of calcium in developing wheat anthers to examine the relationship between Ca2+ and pollen development. During anther development, calcium precipitate formation increased in anther wall cells prior to microspore mother cell meiosis and appeared in microspores, suggesting the presence of a calcium influx from anther wall cells into the locule. Initially, the precipitates in microspore cytoplasm primarily accumulated in the mitochondria and destroyed their inner membranes (cisterns) to become small vacuoles, which expanded and fused, ultimately becoming a large vacuole during microspore vacuolisation. After microspore division and large vacuole decomposition, many calcium precipitates again accumulated in the small vacuoles, indicating that calcium from the large vacuole moved back into the cytoplasm of bicellular pollen.

Migration of sperm cells during pollen tube elongation in Arabidopsis thaliana: behavior during transport, maturation and upon dissociation of male germ unit associations.

The promoter sequence of sperm-expressed gene, PzIPT isolated from the S(vn) (sperm associated with the vegetative nucleus) of Plumbago zeylanica, was fused to a green fluorescent protein (GFP) reporter sequence and transformed into Arabidopsis thaliana to better visualize the live behavior of angiosperm sperm cells. Angiosperm sperm cells are not independently motile, migrating in a unique cell-within-a-cell configuration within the pollen tube. Sperm cells occur in association with the vegetative nucleus forming a male germ unit (MGU). In Arabidopsis, GFP was expressed equally in both sperm cells and was observed using a spinning disk confocal microscope, which allowed long duration observation of cells without bleaching or visible laser radiation damage. Pollen activation is reflected by conspicuous movement of sperm and pollen cytoplasm. Upon pollen germination, sperm cells enter the forming tube and become oriented, typically with a sperm cytoplasmic projection leading the sperm cells in the MGU, which remains intact throughout normal pollen tube elongation. Maturational changes, including vacuolization, general rounding and entry into G2, were observed during in vitro culture. When MGUs were experimentally disrupted by mild temperature elevation, sperm cells no longer tracked the growth of the tube and separated from the MGU, providing critical direct evidence that the MGU is a functional unit required for sperm transmission.

Distribution of calcium in the stigma and style of tobacco during pollen germination and tube elongation.

Potassium antimonate was used to locate loosely bound calcium in the stigma and style of tobacco. The tobacco stigma is wet and covered by a thick layer of glycoprotein exudate at anthesis. The exudate contains abundant vesicles, which are densely labeled with calcium precipitates. When pollen grains arrive at the stigma, become hydrated, and as the pollen swells, Ca(2+) precipitates accumulate at the aperture. Calcium precipitates that accumulate in pollen cytoplasm are initially concentrated within small vacuoles, but as germination proceeds these appear to fuse, forming prominent, densely labeled vesicles that preferentially accumulate near the proximal region of the growing tube. Although the stigma has abundant particles, few calcium precipitates are observed in the transmitting tissue from anthesis to 11 h after pollination. However, at 22 h after pollination, accumulation of calcium increases distally from the stigmatic interface with the transmitting tissue through the length of the style to the ovary. An examination of flowering plants with differing floral biology will be needed to understand the role of loosely bound calcium accumulation and its relationship to tissue-level changes in calcium uptake, maintenance of other calcium pools, including [Ca(2+)](cyt), and in pollen and style maturation during the progamic phase.

[Cytological observation of anther abortion and starch distribution of a cytoplasm male sterile pepper (Capsicum annum L.)].

A cytoplasm male sterile pepper (Capsicum annum L.) was examined using cytochemical method to study its pollen abortion. Thick sections of both anthers of male sterility line 8214A and its maintainer 8214B at different stages were stained using Periodic Acid-Schiff's (PAS) reaction to detect starch distribution. Anther structure and starch distribution in both anthers of male sterility and maintainer line were similar before the meiosis of microspore mother cells. After meiosis, the size of tapetal cells of fertile anthers of maintainer line increased and became high vacuolation. Abundant small starches appeared in the connective cells from tetrad stage to early stage of microspore development. At the late stage of microspore, the tapetal cells began to degenerate and the starches in the connective cells became large. Bi-cellular pollen synthesized starches after the large vacuole of vegetative cell disappeared, and abundant starches were stored in the mature pollen. In the anthers of male sterile line, meiosis of microspore mother could occurred and the tetrads could be formed in the locule, but the tetrads were extruded together because the locule could not enlarge its space. Finally, the tetrad microspores degenerated. The development of vascular tissue of the sterile anthers was normal and abundant starches were stored in the connective cells, which suggested that the function of plant transporting polysaccharide into anther was normal but tapetum could not transport the polysaccharide into locule. According to our result, the pollen abortion occurred in the tetrad stage and the abnormal development of tapetal cells might be the reason which induced tetrad microspore abortion in this male sterile pepper.

[Isolation of sperm cells of Allium tub rosum Roxb].

Pollen grains of Allium tuberosum Roxb broke and released their content including generative cells using osmotic shock method. In a medium containing 0.05% CaCl2, 0.01% Boric acid, 0.01%KH2PO4, 15%PEG 10% sucrose (710 mOsmol/kg H2O) 86% pollen grains germinated and grew out pollen tubes, which broke after transferred into 6% mannitol solution, and released tube content including generative cell. When pollen grains were cultured in the same medium but adding 0.1% casein, a few generative cells divided into two sperm cells. Stigmas of Allium tuberosum Roxb were pollinated at second day after anthesis and the styles grow 3 h in vivo. Then the styles were cut and cultured in a medium for about 6-8 h, some pollen tubes grew out of the cut end of the style. The cut end of the style was transferred into a solution containing 6% mannitol to burst pollen tubes. Pairs of sperm cells of Allium tuberosum Roxb were released and collected using a micromanipulator.

[Isolation of zygotes and apical and basal cells of bicelluare proembryo from Torenia fournieri].

Torenia fournieri is a special plant with embryo sac partly protruding through the micropyle of ovule, and the cells of egg apparatus can be clearly observed using light microscope. Zygotes and the cells of bicellular proembryo could be isolated using enzymatic digestion. In the solution containing 0.05% cellulase and 0.05% pectinase, 14-15 zygotes were isolated from 50 ovules in 1h. In the solution containing 0.2% cellulose, 0.4% hemicellulase and 0.2% pectinase, 19 pairs of apical and basal cells of bicellular proembryo could be isolated from 50 ovules in 1 h. The isolated zygotes and epical and basal cells were collected using a micromanipulator up to a number which will prepare for suing molecular methods to probe the mechanism of early embryogenesis of higher plants.

[Isolation of egg cells from Brugmansia aurea Lagerh "Goildens Kornett"].

Viable egg cells of Brugmansia aurea Lagerh "Goildens Kornett" were isolated using mechanical dissection and enzymic digestion. The ovules were cut from middle part and pushed its micropylar position using a dissection needle. Generally the three cells of egg apparatus were released from cut end of ovule. When the ovules put an isolating solution only containing 0.04% CaCl2, 1% BSA and 12% mannitol, 7 egg apparatus could be isolated from 40 ovules within 2 h. However, it is some difficult to separate egg cell from two synergids. When ovules were first incubated in an enzymic solution containing 1% Pectinase (Serva), 1% Cellulase (Onozuka RS) and 12% mannitol for 30 min, and then transferred into the above-mentioned isolating solution to dissect, 8 egg apparatus could be isolated from 40 ovules within 2h and egg cell is easy to separate from two synergids. The isolated egg cells of Brugmansia aurea Lagerh "Goildens Kornett" could be used in vitro fertilization to explore fertilization mechanism and in egg development using molecular methods.

[Isolation of the alternative oxidase from Arum maculatum].

There is plenty of alternate oxidase (AOX) in the inflorescences of thermogenic A rum maculatum. The isolated mitochondria exhibited a high activity, consuming oxygen on average 32 micromoles/min. The concentration of the isolated protein from mitochondria was 14.0 mg/ml. The isolated mitochondria were crashed by osmotic method to isolate matrix, membrane, and membrane soluble and insoluble proteins. The whole mitochondria, membrane and membrane soluble protein showed AOX activity while the matrix and membrane insoluble protein did not displayed AOX activity. The proteins were purified with FPLC by adding deoxycholamide dBC. The purified enzyme fraction exhibited a high specific activity, which could be kept for at least 6 months when stored at -70 degrees C. Furthermore, we used a silver stain system to identify the AOX, which showed 4 different protein bands from 30 kD to 32 kD. 2-dimensional electrophoresis showed 4 isoelectric points in the range from pH6.4 to 7.4 respectively.

[Studies on the calcium distribution in developing synergids of lettuce (Lactuca sativa L.)].

Potassium antimonite was used to locate calcium in the synergids of lettuce (Lactuca sativa L) during their development. The two synergids on 3d before anthesis formed evident polarity with most cytoplasm located in the micropylar end and nucleus in the middle and a big vacuole in the chalazal end. At this time, calcium precipitates were a few in both cells. Calcium precipitates in the two synergids began to increase on 2d before anthesis. Synergid wall in the micropylar end thickened on 1d before anthesis, in which many calcium precipitates located. Near anthesis, synergids formed filiform apparatus in which abundant calcium precipitates accumulated to prepare for attracting pollen tubes entering. At anthesis, the distribution of calcium precipitates between two synergids was the same. At 1h after pollination, calcium precipitates evidently increased in one synergid that seemed to degenerate, the other one was persistent and the distribution of calcium granules did not change. Two synergids kept intact at 1d after emasculated, and the distribution of calcium precipitates did not display difference, suggesting that the degeneration of one synergid was caused by approaching pollen tubes which might give some signal to induce calcium increase of the synergid. Before fusion of sperm cell with egg cell, the cytoplasm of degenerated synergid embraced the egg and formed a thin layer between the egg and the central cell. Calcium precipitates in the different parts of degenerated synergid were closely connected with the fertilization: calcium precipitates accumulated in the near chalazal end of degenerated synergid at 1h after pollination. At 2.5h after pollination, the calcium precipitates increased at the chalazal end, especially abundant in the thin layer between the egg and the central cell. However, at 4h after pollination, the fertilization had finished at this time, the distribution of calcium precipitates in degenerated synergid changed again: the precipitates decreased at the chalazal end and increased at the micropylar end. The above-mentioned results suggested that calcium in the degenerated synergid played an important role during lettuce fertilization.

[Introduction of hexaploid of Chinese narcissus and analysis of its chromosome change].

Anthers of Chinese narcissus (Narcissus tazetta L. var chinesis Roem) were used as explants for callus induction and plant regeneration. About 80% anthers produced callus and 28% of the callus differentiated out bulbs, making a good experiment system of tissue culture of Chinese narcissus for further cellular and gene engineering. The 700 callus were treated by 0.5% colchicin for 5-6 days and then transformed into a MS medium containing 3 mg/L 6-BA to induce differentiation. 90 bulbs were obtained and 55 bulbs among them were checked the chromosome number from their root tips for three times. 29 bulbs (53%, 29/55) still kept triploidy and the most cells of root tips contained 30 chromosomes. 22 bulbs (40%, 22/55) displayed aneuploidy and the most cells of its root tips contained 10-50 chromosomes. 4 bulbs displayed hexaploidy and contained 60 chromosomes. After three months growing, the cells of root tips containing aneuploidy chromosomes disappeared, and the bulbs became triploidy. The chromosomes of 4 hexaploidy bulbs did not changed during three checks. The origin and disappearance of aneuploidy cells of Chinese narcissus after treated by colchicin were discussed.

[Isolation of sperm cells from rice pollen tubes].

Isolation of sperm cells from higher plants is a basis for studying the mechanism of double fertilization. In this study, the isolation of rice sperm cells from pollen tube was conducted. When fresh pollen grains from nearly blooming flowers were put into a medium containing 20% sucrose, 10% polyethylene glycol 4500 (PEG 4500), 0.05% CaCl2, 0.01% boric acid, over 40% pollen grains germinated and formed a pollen tube. After pollen tubes were transformed into a broken solution containing 8% mannitol, the tubes broke and released tube cytoplasm including two sperm cells. However, both sperm cells were enrapt in the cytoplasm and could not be identified. When 0.5% cellulase and pectinase were added into the broken solution, two sperm cells were released from cytoplasm. Both sperm cells could be collected using micromanipulator. We also tried to isolate sperm cells using in vivo-in vitro method: styles were pollinated and pollen tubes were allowed to grow for 40 min in vivo. Then styles were cut near ovary and floated in the same medium above-mentioned for 1 h until tubes emerged from the cut end. The styles with pollen tube were transformed into the broken solution and released the content including two sperm cells.

[The distribution of ATPase during the pollen development of Allium cepa L].

The distribution of ATPase in pollen of Allium cepa L. was studied using Pb3 precipitation technique during pollen development. Only some ATPase precipitates were located in the nucleus of microspore mother cells (MMC) and a few in its cytoplasm. After meiosis of MMC,many ATPase precipitates appeared in the exine of pollen wall of microspore even it was in tetrad, suggesting that ATPase from tapetum is necessary during pollen wall construction. The intine of pollen wall of microspore was synthesized at its late stage and consisted of cellular material which was from microspore. There were also many ATPase precipitates in intine,and the ATPase came from microspore. Then ATPase precipitates in vegetative cell increased and that in generative cell decreased during the development of 2-cellular pollen,suggesting the differentiation of vigor between both cells. The physiological functions of ATPase in developing pollen of Allium cepa L. were analyzed.

[Anther development and sperm isolation of Pseudostellaria heterophylla (Miq.)].

Anther wall is general and tapetum is glandular. The process of meiosis of microspore mother cells is simultaneous and the tetrads are tetrahedral. The mature pollen of Pseudostellaria heterophylla (Miq.) is tree-celled. There are 22-30 germ pores on the pollen wall. Many pollen grains could burst in 10% mannitol or 15% sucrose solution and release a pair of sperm cells which could keep alive for 25-50 min by FDA fluorescence. Using micromanipulator the released sperm cells could be collected. When pollen grains were put into a solution containing 0.03% CaCl2, 0.01% H3BO3, 0.01% KH2PO4 and 20% PEG for 2-5 min, they would germinate and the pollen tubes would reach 815 microm at 2h after cultured. A pair of sperms would enter into pollen tube when it grew to 500-600 microm. The fluorescence of both sperms would be observed clearly in pollen tube after DAPI staining. When the pollen tubes were burst in a bursting solution, a pair of sperms would be released from pollen tube.

[Cytochemical observation on the developing anthers of Allium cepa L].

Polysaccharides was detected using the periodic-acid-Schiff's (PAS) technique and lipid detected using Sudan black during the anther development of Allium cepa L. Before the meiosis of microspore mother cells there were a few lipid drops in endothecium cells and little polysaccharides in tapetal cells which did not differentiate completely in young anthers. At the stage of tetrad there were still few polysaccharides and lipid material in young anther, and only cell wall of anther wall and callose wall of tetrads displayed the feature of polysaccharids. The size of tapetal cells began to increase at this stage. During microspore development the tapetal cells reached its maximal size, and many lipid drops were accumulated in the cells. However, few lipid drops and starches appeared in microspores. At early stage of 2-celled pollen, the vegetative cell of 2-celled pollen began to accumulate starches. Tapetal cells degenerated at this stage and its lipid drops concentrated to form lipid block. Then the starches in 2-celled pollen disappeared with pollen development, and many lipid drops were accumulated in vegetative cell of nearly mature pollen.

Calcium function and distribution during fertilization in angiosperms.

Calcium has an essential signaling, physiological, and regulatory role during sexual reproduction in flowering plants; elevation of calcium amounts is an accurate predictor of plant fertility. Calcium is present in three forms: (1) covalently bound calcium, (2) loosely bound calcium typically associated with fixed and mobile anions (ionic bonding); and (3) cytosolic free calcium-an important secondary messenger in cell signaling. Pollen often requires calcium for germination. Pollen tube elongation typically relies on external calcium stores in the pistil. Calcium establishes polarity of the pollen tube and forms a basis for pulsatory growth. Applying calcium on the tip may alter the axis; thus calcium may have a role in determining the directionality of tube elongation. In the ovary and ovule, an abundance of calcium signals receptivity, provides essential mineral nutrition, and guides the pollen tube in some plants. Calcium patterns in the embryo sac also correspond to synergid receptivity, reflecting programmed cell death in one synergid cell that triggers degeneration and prepares this cell to receive the pollen tube. Male gametes are released in the synergid, and fusion of the gametes requires calcium, according to in vitro fertilization studies. Fusion of plant gametes in vitro triggers calcium oscillations evident in both the zygote and primary endosperm during double fertilization that are similar to those in animals.