Fluorophore-conjugated lectin labeling of the cell surface of isolated male and female gametes,central cells and synergids before and after fertilization in maize

Three fluorescein isothiocyanate (FITC)-conjugated lectins, Canavalia ensiformis agglutinin (Con A), Triticum vulgaris agglutinin (WGA) and Phaseolus vulgaris erythroagglutinin (PHA-E), were used as probes to localize sugar moieties of glycoconjugates on the cell surface of isolated maize sperm, egg, central, antipodal cells, synergids, and in vitro- and in vivo-fertilized zygotes. Fluorescence signals on the surface of the cells were due to specific binding. Calcium was necessary for WGA and PHA-E binding and enhanced Con A labeling. Differences in glycoconjugate composition of the membranes of gametes and other embryo sac component cells were found. FITC-Con A strongly labeled egg and central cells, but labeled sperm only weakly. FITC-WGA binding sites were detected on egg, but not sperm cells. Con A and WGA binding sites were equally distributed around egg and central cell protoplasts. FITC-PHA-E binding sites were not found on sperm and egg cells before fertilization. Binding sites of these lectins were located on synergids, especially on their filiform apparatus. Interestingly, WGA binding to egg cells was enhanced after fertilization, whereas PHA-E binding to egg cell membranes could only be detected after fertilization. These results suggest the occurrence of fertilization-induced changes in glycoconjugate composition of the maize egg cell membrane. An increase in the number of WGA and PHA-E binding sites was also observed on newly formed cell walls of cultured two-celled embryos derived from in vitro-produced zygotes.     

Comprehensive transcriptome analysis of phytohormone biosynthesis and signaling genes in microspore/pollen and tapetum of rice

To investigate the involvement of phytohormones during rice microspore/pollen (MS/POL) development, endogenous levels of IAA, gibberellins (GAs), cytokinins (CKs) and abscisic acid (ABA) in the mature anther were analyzed. We also analyzed the global expression profiles of genes related to seven phytohormones, namely auxin, GAs, CKs, brassinosteroids, ethylene, ABA and jasmonic acids, in MS/POL and tapetum (TAP) using a 44K microarray combined with a laser microdissection technique (LM-array analysis). IAA and GA(4) accumulated in a much higher amount in the mature anther compared with the other tissues, while CKs and ABA did not. LM-array analysis revealed that sets of genes required for IAA and GA synthesis were coordinately expressed during the later stages of MS/POL development, suggesting that these genes are responsible for the massive accumulation of IAA and GA(4) in the mature anther. In contrast, genes for GA signaling were preferentially expressed during the early developmental stages of MS/POL and throughout TAP development, while their expression was down-regulated at the later stages of MS/POL development. In the case of auxin signaling genes, such mirror-imaged expression observed in GA synthesis and signaling genes was not observed. IAA receptor genes were mostly expressed during the late stages of MS/POL development, and various sets of AUX/IAA and ARF genes were expressed during the different stages of MS/POL or TAP development. Such cell type-specific expression profiles of phytohormone biosynthesis and signaling genes demonstrate the validity and importance of analyzing the expression of phytohormone-related genes in individual cell types independently of other cells/tissues.     

南方红豆杉种子综合处理过程中内源激素的动态变化

为揭示南方红豆杉种子内源激素与休眠的关系,采用酶联免疫吸附法(ELISA)测定了经过层积处理的种皮和胚乳的脱落酸(ABA)、赤霉素(GA3 )、吲哚乙酸(IAA)、玉米素核苷(ZR)4种内源激素含量的变化情况.结果表明:种子胚乳中内源ABA的含量随着层积时间的延长而逐渐下降,GA含量增加,IAA和ZR的含量先增加后降低...     

棉花胚珠纤维发育的研究

将未受精的棉花胚珠漂浮培养在加有不同植物激素的BT培养基上,培养48小时或96小时后,用扫描电镜观察纤维发育情况,以及测定胚珠内IAA氧化酶活性变化及内源ABA的含量变化,并和同一时期的大田生长的胚珠进行比较。结果表明:IAA+GA_3是最佳激素组合。在这种激素组合的培养基中培养的未受精胚珠,在纤维发育、酶活性变化等方面,均与大田生长的胚珠相似。这一激素组合还能抑制离体胚珠内源ABA的增长,但同一时期的大田生长的胚珠,其内源ABA含量却相对要高。     

Localization of arabinogalactan proteins in egg cells, zygotes, and two-celled proembryos and effects of beta-D-glucosyl Yariv reagent on egg cell fertilization and zygote division in Nicotiana tabacum L

Arabinogalactan proteins (AGPs) have been implicated in a variety of plant development processes including sexual plant reproduction. As a crucial developmental event, plant sexual reproduction generally occurs inside an ovule embedded in an ovary. The inaccessibility of the egg cells, zygotes, and embryos has hindered our understanding of the importance of AGPs in the early events involving fertilization, zygotic division, and early embryogenesis. In this study, the well-established in vitro zygote and ovary culture systems, together with immunofluorescence and immunogold labelling techniques, were employed to investigate the role of AGPs in the early events of sexual reproduction in Nicotiana tabacum. Dramatic changes in AGP content during ovule development were evidenced by western blotting. Subcellular localization revealed that AGPs are localized in the plasma membrane, cell wall, and cytoplasm of pre- and post-fertilized egg cells, and cytoplasm and vacuoles of two-celled proembryos. Abundant AGPs were detected in unfertilized egg cells; however, the level of AGPs substantially decreased in fertilized egg cells. Polar distribution of AGPs in elongated zygotes was observed. The early two-celled proembryos just from zygote division displayed accumulation of AGPs at a low level, while in the elongated two-celled proembryos at the late stage, the AGP content clearly increased. Provision of betaGlcY, a synthetic phenylglycoside that specifically binds AGPs, to the in vitro cultures of isolated zygote and fertilized ovaries increased abnormal symmetrical division of zygotes. In the culture of pollinated but unfertilized ovaries, addition of betaGlcY resulted in arrest of fertilization of the egg cells, but had no effect on fertilization of the central cells. The possible roles of AGPs in fertilization, zygotic division, and proembryo development are discussed.     

γ-Irradiation of Barley Seeds and Its Effect on the Phytohormonal Status of Seedlings

We investigated the effect of γ-irradiation (4–50 Gy) of barley seeds (Hordeum vulgare L., cv. Nur) on the content of endogenous phytohormones–stimulators of plant growth and development: indol-3-acetic acid (IAA), indolyl-3-butyric acid (IBA), zeatin and abscisic acid (ABA). The ratio (IAA + IBA + zeatin)/ABA from the third to the seventh day of germination has been measured. It was shown that the changes in the content of phytohormones as a function of the radiation dose were nonlinear. In the dose range of 4–20 Gy, phytohormones balance was changed due to increased content of growth stimulators and decreased ABA content. Using a dose of 50 Gy led primarily to a decrease in the content of growth stimulators and an increase in ABA content, and the ratio (IAA + IBA + zeatin)/ABA shifted toward ABA content.     

Isolation and in vitro culture of zygotes and central cells of Oryza sativa L.

 All component cells of the embryo-sac before and after fertilization in rice were isolated by manual microdissection under conditions either free of enzymes or combined with a short pulse of enzymatic treatment.In general, the frequency of isolated unfertilized or fertilized egg cells or central cells reached 15–40%. Various component cells of the embryo-sac after isolation were distinguished by their own morphological characteristics. The isolated cells were cultured in a microchamber and fed with dividing rice suspension cells. Both unfertilized and fertilized egg cells and central cells were induced to divide. Among them only the fertilized egg cells (the zygotes) developed into proembryo-like multicellular structures. The frequency of the first zygotic division and the frequency of multicellular structures were higher using the non-enzymatic method than using the enzymatic one. Received: 14 January 1999 / Revision received: 25 March 1999 / Accepted: 17 April 1999     

Ultrastructural Observations on Parthenogenesis and Antipodal Apogamy of Allium tuberosum Roxb

Alliurn tuberosum Roxb is a species characterized by spontaneous parthenogenesis and antipodal apogamy. This paper deals with the ultrastructural changes during these processes. Before pollination, the mature egg cell contained abundant mitochondria with well developed cristae, spherical or ellipsoidal plastids and some polyribosomes, which suggested a relatively high metabolic activity. After fertilization, in zygotes the mitochondria changed to irregular shape and their cristae degenerated, the plastids elongated or became cup-shaped, the polyribosomes decreased and the free ribosomes increased in number. Some unfertilized egg ceils, two days after anthesis, showed similar ultrastructural changes as those taking place in zygote, which. seemed to be a sign of triggering to parthenogensis. In Allium tuberosum Roxb, the three antipodai cells bore a close resemblance to the egg apparatus: Among them two antipodal cells were similar to the synergids with a filiform apparatus-like structure and plentiful organelles at their chalazal end. The other was an egg-like antipodal cell which could undergo similar ultrastructural changes as those happened in zygote, leading to divide into apogamic proembryos two days after anthesis. The problems of parthenogenesis and antipodal apogamy in Allium tuberosum Roxb is diicussed in view of ultrastructural features of the egg and egg-like antipodal cell.     

Calcium changes in ovules and embryo sacs of Plumbago zeylanica L.

Calcium was localized in ovules of Plumbago zeylanica from 1 day before anthesis to 3 days after anthesis using potassium antimonate and transmission electron microscopy in pollinated and emasculated flowers. At 1 day before anthesis, embryo sacs (containing an egg cell, a central cell and zero to three accessory cells) appear mature and contain abundant calcium precipitates (ppts), in contrast to nucellar cells. At anthesis, the vacuoles of nucellar cells have enlarged, and micropylar cells, in particular, are heavily labeled with calcium ppts. As pollen tubes elongate through ovular tissues, ppts diminish in ovular cells and become concentrated in the pollen tube cell wall. After fertilization, the calcium ppts sharply diminish in fertilized ovules; in unfertilized ovules, calcium ppts remain abundant up to 3 days after anthesis (when unfertilized ovules are shed). The distribution of calcium in the ovule changes in apparent response to fertilization, suggesting that calcium content may be related to the attraction and receipt of the pollen tube. In contrast with conventionally-organized embryo sacs with synergids, Plumbago accumulates calcium in the egg cell. Received: 30 December 1999 / Revision accepted: 24 March 2000     

Adaptation of chickpea to desiccation stress is enhanced by symbiotic rhizobia

This study examined the influence of three inoculant strains of Bradyrhizobium japonicum (Thal-8, Tal 620, Dulawala) on the ability of chickpea (Cicer arietinum (L.) to adapt to drought-stress. Strain Thal-8 was most effective in the root-nodule symbiosis and also partially alleviated decreased growth and yield imposed by drought stress. Strain Thal-8, in pure culture, also produced higher amounts of gibberellic acid (GA) and indole-3-acetic acid (IAA) and lower amounts of abscisic acid (ABA) than the other two test strains. Thal-8 increased the root biomass, GA and IAA contents of leaves of chickpea plants, including ICC 4948NN, a non-nodulating line. These results are consistent with the hypothesis that GA and IAA is produced by the Thal-8 strain and/or elevates levels of these phytohormones in chickpeas. This contributes to its high performance as a nitrogen-fixing microsymbiont. The growth-promoting response evoked by different strains of Bradyrhizobium correlated with higher ratios of GA and IAA relative to ABA phytohormones in the plants.     

强旱生小灌木绵刺劈裂生长过程中内源激素含量变化的研究

 研究强旱生小灌木绵刺(Potaninia mongolica)劈裂生长过程中内源激素含量的变化。结果表明：1）4种生长状态中,完全劈裂的植株的叶片及劈裂发生部位ABA的含量比其它3种状态的都低,而其根中ABA的含量最大。同其它几种激素相比,ABA在绵刺体内的含量最大;2）劈裂生长发生之前,在劈裂发生部位IAA积累量大,尤其是在即将劈裂的过渡植株的劈裂发生部位IAA含量最大;3）劈裂生长发生过程中GA3含量的变化与IAA的变化有同步性;4）ZR的含量也是在劈裂生长发生前的绵刺的劈裂发生部位中较大,随着劈裂生长的发生,植物从根部向叶片及劈裂发生部位运输的ZR有逐渐降低的趋势,而在劈裂生长发生的过渡阶段,ZR从根部向劈裂发生部位运输的比例较大,分别为19.44%和20%;5）IAA、GA3、ZR 三者协调促进劈裂发生部位细胞的生长和分裂,而ABA的积累对绵刺适应干旱的环境条件起到了一定的调节作用。     

高效液相色谱法同时检测棉花根中的多种植物激素

目的：探寻高效液相色谱同时检测棉花根中多种植物激素含量的方法。方法：采用WatersC18反相色谱柱（4.6×250mm,5μm）,在柱温为35℃、流速为1mL.min-1的条件下,以乙腈和三乙胺溶液为流动相梯度洗脱,在每种物质的保留时间附近切换至最大吸收峰（GA3除外）波长作为检测波长,并与254nm同一波长检测多种植物激素含量的方法进行比较,分离和检测棉花根中的玉米素（Z）、玉米素核苷（ZR）、赤霉酸（GA3）、生长素（IAA）和脱落酸（ABA）的含量。结果：切换波长法检测5种植物激素的灵敏度和回收率均较高,检出限均较低。回收率为：Z 96.82%、ZR 94.14%、GA3 92.75%、IAA 93.38%、ABA 95.57%;检出限为：Z 0.1μg.mL-1、ZR 0.1μg.mL-1、GA3 0.5μg.mL-1、IAA 0.3μg.mL-1;ABA 0.05μg.mL-1,能准确检测出棉花根中Z、ZR、GA3、IAA和ABA的含量。结论：采用Waters C18反相色谱柱（4.6×250mm,5μm）,在柱温为35℃、流速为1mL.min-1的条件下,以乙腈和三乙胺溶液为流动相梯度洗脱,结合切换波长法能同时检测出植物组织中多种植物激素含量。     

莴苣胚囊细胞分离

用酶解和解剖方法分离了莴苣的卵细胞,助细胞,中央细胞和合子。莴苣子房先在酶液中酶解40～50min,然后在不含酶的分离液中用解剖针解剖子房。在解剖出的胚囊中,可看到卵细胞,两个助细胞和中央细胞的轮廓。将胚囊的合点端切破,轻轻挤压胚囊的珠孔端,四个细胞即可逸出。在最佳条件下,90min可从40个子房中分离出29个胚囊,进一步从中分离出11个卵细胞。分离出的胚囊细胞用显微操作仪收集备用。莴苣卵细胞的成功分离为进行离体受精探索创造了条件。     

Simultaneous quantitation of indole 3-acetic Acid and abscisic Acid in small samples of plant tissue by gas chromatography/mass spectrometry/selected ion monitoring

Indole 3-acetic acid (IAA) was analyzed in apple, orange, and prune tissue by GC-MS by monitoring the protonated molecular ion of its methyl ester at mass to charge ratio (m/z) 190 together with the major fragment ion at m/z 130 and the corresponding ions from the methyl esters of either [²H₄]IAA (m/z 194, 134) or [²H₅]IAA (m/z 195, 135). Abscisic acid (ABA) was analyzed by monitoring the major fragment ions of its methyl ester at m/z 261 and m/z 247 and the corresponding ions from the methyl ester of [²H₃]ABA (m/z 264, 250). Detection limits for IAA and ABA were 1 and 10 picograms, respectively using standards and 1 nanogram per gram dry weight for both phytohormones in plant tissue.     

Ovule,Female and Male Gametophyte and Fertilization of Kingdonia uniflora Balfour F. et W. W. Smith

The structure of ovule, female and male gametophyte, double fertilization and the distrubution of starch grains during the fertilization have been studied. The main results are as follows: ( 1 ) Ovule The ovule is anatropous, unitegmic and tenuinucellate. The nucetlus appears cylindric, since megaspores and embryo sac development, its internal cells of nucellus become disorganized, so that only a single layer of epidermal cells remains toward the side of the micropyle, On the other hand, the integument is not as long as nucellus, as a result micropyle is not formed. And no vascular bundle is found in the integument. (2) Female gametophyte The mature embryo sac is slender and is composed of an egg cell, two synergids, a central cell and three antipodal cells. The egg cell is situated slightly away from the tip of embryo sac. Some of them contain starch grains. Synergids occupy the tip of embryo sac. Its wall at micropylar region appears irregular in thickenes and irregular in ingrowths to form the filiform apparatus. The centrateell is very large, and strongly vacuolated Two polar nuclei come to contact closely with each other, but not fuse, or to fuse into a large secondary nucleus before fertilization. The polar nuclei or the secondary nucleus are usually situated at the middle-lower position of the central cell or nearer to the chalazal end above the antipodal cell. It is different from egg cell, no starch grains are found here. In most embryo sacs three antipodal cells are found. They are not as large as those in other plants of Ranunculaceae. But six antipodal cells or the antipodal cell with two nuclei may rarely be found. Like synergid, the wall of them appears not only irregularly thickened, but clearly with irregular ingrowths. In a few antipodal cells the starch garins are usually found near the nucleus. By the end of fertilization, antipodal cells become disintegrated. (3) Male gametophyte Most pollen grains are two-celled when shedding, and rich in starch grains. A few of them contain single nucleus or three-celled. (4) The double fertilization The fertilization of Kingdonia unifiora Balfour f. et W, W. Smith is wholly similar to some plants of Ranunculaceae studied. First, the pollen tube penetrates a degenerating synergid. And the pollen tube discharges its contents with two sperm nuclei into the degenerating synergid cell. One of the two sperms fuses with the nucleus of the egg, and the other fuses with two polar nuclei or the secondary nucleus of the central cell. If one sperm nucleus at first fuses with one of the polar nuclei, and then the fertilized polar nuclei again fuses with other polar nucleus. Secondly, the fertilization of the polar nuclei or the secondary nuclei completes earlier than that of the egg. The primary endosperm nucleus begins to divide earlier than the zygote. It seems that one of the sperm nuclei come to contact with egg nucleus, the other has already fused with polar nuclei or the secondary nucleus. The zygote with a single nucleolus appears until the endosperm with 16–20 cell. Thirdly, before and after fertilization there are one to some small nucleoli in egg nucleus and polar nuclei or secondary nucleus. However they increase in quantity from the beginning of the fusion of male nucleis. These nucleoli quite differ from male nucleoli by their small size, and most of them disappear at the end of fertilization. It may be concluded that the small nucleoli increase in quantity is related to the fusion of male and female nuclei. In the duration of fertilization, in ovule starch distribution is in the basal region of integument. But in embryo sac, onlysome egg cells, or zygotes contain starch grains, a part of which was brought in by pollen tube. Sometimes the starch grains are found in some synergids and antipodal cells. No starch grains are found in the central cell.     

Electrofusion-mediated transmission of cytoplasmic organelles through the in vitro fertilization process,fusion of sperm cells with synergids and central cells,and cell reconstitution in maize

Summary Fusion products were created by the electrofusion of single sperm cells with single synergids and central cells. The synergid was also fused with the sperm cell, occasionally in the presence of adhering second synergids, egg cells, and central cells. Single egg cells were fused with single sperm cells in the presence of adhering synergids and the central cell. Cytoplasmic organelles were transmitted through the fertilization process by electrofusion using cytoplasts of maize mesophyll cells. Cell reconstitution was achieved by fusion of one or two sperm cells with single enucleated protoplasts, thus creating a haploid or a diploid cell.