拟南芥RabD2b蛋白氨基酸突变对定位和功能的影响

Rab蛋白4个保守的鸟嘌呤核苷酸结合结构域G1、G3、G4和G5共同参与了与GTP的结合及水解过程。将拟南芥(Arabidopsis thaliana)RabD2b(AtRabD2b)G4结构域的重要氨基酸位点天冬酰胺(asparagine, N)突变为异亮氨酸(isoleucine, I)(AtRabD2b^[N121I]), 并研究了N121I突变对AtRabD2b亚细胞定位和功能的影响。结果表明, N121I突变使AtRabD2b由原来的高尔基体点状定位转变为高尔基体和细胞质弥散定位。AtRabD2b能够互补酿酒酵母(Saccharomyces cerevisiae)同源蛋白Ypt1突变产生的功能缺陷, 而AtRabD2b^[N121I]仅能部分互补Ypt1的功能。AtRabD2b^[N121I]转基因植株表现出矮化、丛生、不育和茎顶端坏死等多效性异常表型, 与AtRabD2b转基因植株出现的主茎异常抽出表型不同。上述结果表明, N121I突变不仅引起了AtRabD2b亚细胞定位的改变, 而且影响了其正常功能。     

欧石楠体细胞胚发生和植株再生

以欧石楠茎段为外植体,研究其体细胞胚胎发生和植株再生。对影响茎段不定芽分化及胚性愈伤组织诱导的主导因子进行比较分析,并研究其体胚萌发、生根及移栽;同时,采用树脂切片法对茎段脱分化产生胚性愈伤组织及体胚发育过程进行组织细胞学观察。结果表明,接种在1／2WPM基本培养基上的茎段,胚性愈伤组织诱导率为88．7％,显著高于其他处理,不定芽诱导率可达90．6％,平均分化倍数为3．6个,平均分化苗高3．82cm;体细胞经过成熟培养后。在添加1．0mg·L-1 ZT和0．3mg·L-1 IBA的1／2WPM培养基上萌发,萌发的体胚在I／2WPM附加0．2mg·L-1 NAA和0．3mg·L-1 IBA的培养基上形成完整的体胚苗植株,体胚苗生根率达到87．4％,经炼苗后移栽到蛭石：珍珠岩=3：1（V／V）的栽培基质中,成活率可达63．7％。在显微镜下可观察到球形胚、心形胚、鱼雷形胚和子叶形胚;体细胞胚以间接方式发生,表现为愈伤组织外层细胞直接发生和愈伤组织组织内部细胞发生。     

木本植物非均质化组织石蜡切片制作方法

在常规石蜡切片技术的基础上, 针对木本植物茎段木质化程度高、硬度大以及各部分组织硬度不均匀等特点, 选取核桃(Juglans regia)茎段以及芽接愈合区域组织为实验材料, 对固定、软化和脱水等关键步骤进行改进, 获得结构完整且染色清晰的茎段组织和嫁接愈合区域组织切片, 可清晰地观察到各部分组织的形态特征和愈合过程中的发育特征, 且缩短了制片周期。采用改良后的实验流程成功获得了苹果(Malus pumila)、桃(Amygdalus persica)、杏(Armeniaca vulgaris)、李(Prunus salicina)和杨(Populus tomentosa)的茎段横截面切片。该方法为从解剖学上研究林木茎段生长机制和形态发育变化提供技术基础, 为非均质化植物材料的石蜡切片制作提供参考。     

光周期对西葫芦185品系顶芽和叶片衰老的调控

在短日照下 ,西葫芦 (CucurbitapepoLinn .) 185品系的植株发生衰老。结构学、基因表达与系列生化分析证实 :短日照启动了顶端分生组织由营养生长锥向花芽的转化 ,进而其组成细胞发生编程性死亡 (PCD) ,导致顶端生长势的丧失 ;与长日照处理相比 ,短日照处理在发育晚期也引起大量叶肉细胞发生PCD ,进而叶片出现衰老。核酸酶活性的高度表达是PCD过程中一个非常重要的分子事件。实验证实 ,西葫芦 185品系植株衰老进程的发生与顶端分生组织和叶肉细胞中发生PCD密切相关。     

三叶橡胶花粉植株的诱导

从三叶橡胶花药培养获得5株花粉植株。它们都具有主根、轮生侧根、茎、子叶、第一对真叶和顶芽。在显微镜下观察到花粉粒发育成为胚状体的全过程。对15个直径2—3毫米的胚状体所进行的细胞学观察表明,它们都是单倍体(染色体数为18)。小植株根尖细胞染色体观察表明,除少数仍是单倍体细胞外,还观察到大量非整倍体细胞。未见到有双倍体细胞。证明用花药培养所得植株来源于花粉。     

光周期对西葫芦185品系顶芽和叶片衰老的调控

在短日照下,西葫芦(Cucurbita pepo Linn.)185品系的植株发生衰老.结构学、基因表达与系列生化分析证实:短日照启动了顶端分生组织由营养生长锥向花芽的转化,进而其组成细胞发生编程性死亡(PCD),导致顶端生长势的丧失;与长日照处理相比,短日照处理在发育晚期也引起大量叶肉细胞发生PCD,进而叶片出现衰老.核酸酶活性的高度表达是PCD过程中一个非常重要的分子事件.实验证实,西葫芦185品系植株衰老进程的发生与顶端分生组织和叶肉细胞中发生PCD密切相关.     

冰冻切片技术在海滨锦葵细胞学研究中的应用

为解决普通石蜡切片观察植物样品繁琐与耗时长的问题,利用改进的蔗糖保护—液氮速冻切片法,进行海滨锦葵不同器官细胞学研究。结果表明:(1)适合于海滨锦葵不同器官的最适蔗糖浓度不同,适于含水量较大的营养器官(根、茎和叶)的最适蔗糖浓度比含水量较小的花器官(柱头裂片、花柱、子房和花药)高。(2)利用最适蔗糖浓度的蔗糖保护-液氮速冻切片方法,获得了完整而清晰的海滨锦葵各器官组织细胞结构的切片;海滨锦葵具有典型的双子叶草本植物营养器官的组织细胞结构特征,花柱为闭合型花柱,子房为多室复子房。表明基于蔗糖保护-液氮速冻的冰冻切片技术在植物细胞学研究中具有广阔的应用前景。     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

不同茎径甘蔗品种茎尖原生分生组织不同区域细胞分裂频率差异

为探明甘蔗茎尖原生分生组织不同区域细胞分裂频率差异及其与茎径和株高的关系,对6个不同茎径品种5个不同生长时期的甘蔗茎尖进行石蜡连续纵切片显微观察,发现甘蔗茎尖原生分生组织各区域细胞分裂频率有明显差异:周缘分生区细胞(3.89%)原体原始细胞区(2.67%)髓分生区(1.46%)原套原始细胞区(1.30%),以上差异均达到显著水平;各区域细胞分裂频率与甘蔗茎径均呈正相关,其中髓分生区和原套原始细胞区细胞分裂频率与茎径的相关系数较大,分别为r~2=0.856*、r~2=0.925*;各区域细胞分裂频率与甘蔗株高均呈负相关,其中原体原始细胞区细胞分裂频率与株高相关系数r~2=-0.728*。结果表明对原生分生组织各区域细胞分裂频率的精确量化,可以揭示甘蔗茎尖原生分生组织各区细胞与其特征的内在联系和不同区域细胞活动能力差异是甘蔗茎增粗的细胞学基础。     

Dynamic reorganization of microtubules and microfilaments in flax cells during the resistance response to flax rust infection

The cytoskeleton in plant cells is a dynamic structure that can rapidly respond to extracellular stimuli. Alteration of the organization of microtubules and actin microfilaments was examined in mesophyll cells of flax, Linum usitatissimum L., during attempted infection by the flax rust fungus, Melampsora lini (Ehrenb.) Lev. Flax leaves that had been inoculated with either a compatible (yielding a susceptible reaction) or an incompatible (yielding a resistant reaction) strain of M. lini were embedded in butyl-methylmethacrylate resin; sections of this material were immunofluorescently labelled with anti-tubulin or anti-actin and examined using confocal laser scanning microscopy. In uninfected leaves, microtubules in the mesophyll cells formed a transverse array in the cell cortex. Microfilaments radiated through the cytoplasm from the nucleus. In an incompatible interaction, microtubules and microfilaments were extensively reorganized in mesophyll cells that were in contact with fungal infection hyphae or haustorial mother cells before penetration of the cell by the infection peg. After the initiation of haustorium development, microtubules disappeared from the infected cells, and growth of the haustoria ceased. In an incompatible interaction, hypersensitive cell death occurred in more than 70% of infected cells but occurred in less than 20% of cells in compatible interactions. After the infected cell had undergone hypersensitive cell death, the cytoskeleton in neighbouring cells became focused on the walls shared with the necrotic cell. In compatible interactions, reorganization of the cytoskeleton was either not observed at all or was observed much less frequently up to 48 h after inoculation.Abbreviations FITC fluorescein isothiocyanate - WGA wheatgerm agglutinin We thank Dr. G.J. Lawrence for providing valuable discussions and materials.     

Starvation induces apoptosis in the midgut nidi of <Emphasis Type="Italic">Periplaneta americana</Emphasis>: a histochemical and ultrastructural study

The effects of starvation on cell death in the midgut of Periplaneta americana were studied histochemically and ultrastructurally. TUNEL assays showed that cell death began to increase in the columnar cells and nidi, the nests of stem cells and newborn cells from 2 weeks of starvation. A significant increase in cell death occurred in the nidi after 4 weeks of starvation. Cockroaches starved for 4 weeks showed active-caspase-3-like immuno-reactivity both in the columnar cells and nidi, whereas control cockroaches that were fed for 4 weeks showed this reactivity only in the apical cytoplasm of columnar cells. Electron microscopy revealed no chromatin condensation in the nucleus of columnar cells of cockroaches, whether fed or starved for 4 weeks. Starved cockroaches exhibited many small vacuoles in the cytoplasm of some columnar cells and “floating” organelles including nuclei in the lumen. A 4-week starvation induced the appearance of cytoplasmic fragmentation and secondary lysosomes in the nidi. Each fragment contained nuclear derivatives with condensed chromatin, i.e. apoptotic bodies. Mitotic cells were found in some, but not all nidi, even within the same starved sample. Fragmentation was not observed in the nidi of control cockroaches. Thus, starvation increases cell death not only in the columnar cells, but also in the nidi. The cell death in the nidi is presumably apoptosis executed by caspase 3.     

Cell elongation and cell division in elongating lettuce hypocotyl sections

The roles of cell division and cell elongation in the growth of sections excised from hypocotyls of lettuce (Lactuca sativa L. cv. Arctic) were investigated. Elongation of sections incubated in the light is inhibited compared to dark-grown sections and this inhibition is reversed by gibberellic acid (GA₃). The elongation of both dark-grown and GA₃-treated, light-grown sections can be enhanced by 10mM KCl. Under all conditions of incubation, elongation growth is greatest in the uppermost quarter of the hypocotyl section while the basal quarter does not elongate. In darkness the two apical segments of sections marked into four equal parts grow at the same rate, while in light, growth of the apical segment exceeds that of the second segment. Cell division in cortical or epidermal cells, as measured by mitotic index or cell number, is not affected by illumination conditions nor by GA₃ or KCl treatments. Although -irradiation and FUDR pretreatment eliminate or cause a marked reduction in cell division in the excised hypocotyl, sections from seeds irradiated with -rays or incubated in 5-fluorodeoxyuridine elongate in response to GA₃ and KCl treatment as do sections from non-pretreated controls. Therefore, since neither GA₃ nor darkness affect celldivision activity and since treatments which eliminate or significantly reduce cell division do not affect growth, we conclude that the effect of GA₃ and darkness in this material is to increase cell elongation.Abbreviations FUDR 5-fluorodeoxyuridine - GA(s) gibberellin(s) - GA₃ gibberellic acid     

Ultrastructural analysis of the behavior of the dimorphic fungus Microbotryum violaceum in fungus-induced anthers of female Silene latifolia flowers

Summary. The development of male organs is induced in female flowers of the dioecious plant Silene latifolia by infection with the fungus Microbotryum violaceum. Stamens in a healthy female flower grow only to stage 6, whereas those in an infected female flower develop to the mature stage (stage 12), at which the stamens are filled with fungal teliospores instead of pollen grains. To investigate these host–parasite interactions, young floral buds and fungus-induced anthers of infected female flowers were examined by electron microscopy following fixation by a high-pressure freezing method. Using this approach, we found that parasitic hyphae of this fungus contain several extracellular vesicles and have a consistent appearance up to stage 8. At that stage, parasitic hyphae are observed adjacent to dying sporogenous cells in the infected female anther. At stage 9, an increased number of dead and dying sporogenous cells is observed, among which the sporogenous hyphae of the fungus develop and form initial teliospores. Several types of electron-dense material are present in proximity to some fungi at this stage. The initial teliospores contain two types of vacuoles, and the fungus cell wall contains abundant carbohydrate, as revealed by silver protein staining. The sporogenous cell is probably sensitive to infection by the fungus, resulting in disruption. In addition, the fungus accelerates cell death in the anther and utilizes constituents of the dead host cell to form the mature teliospore. Correspondence and reprints (present address): Molecular Membrane Biology Laboratory, RIKEN, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan.     

An electron microscopic study of the salivary gland of Rhynchosciara angelae

Summary The structure of the salivary gland of the dipteran insect Rhynchosciara angelae in a defined stage of the larval development, characterized by the synthesis and storage of secretion product, is described. Observations were made with both Nomarski optics and electron microscopy. Filiform projections extending into the lumen of the gland were observed in the apical portion of the cells. At the basal region junctions, characterized as hemidesmosomes, were observed between the membrane of the cell and the basal lamina. The plasma membrane presents numerous infoldings into the cell increasing considerably the surface area at this region. Throughout the cytoplasm of the gland cells numerous mitochondria, Golgi complexes, microtubules, profiles of endoplasmic reticulum, secretion granules and glycogen granules were observed. Carbohydrates were detected on ultrathin sections by using the periodic acid-silver methenamine and the periodic acid-thiosemicarbazide-silver proteinate techniques.     

An attachment tip and pili-like structures in insect- and plant-pathogenic spiroplasmas of the class Mollicutes

Ultrastructural studies using scanning electron microscopy (SEM), negative-staining transmission electron microscopy (TEM), and thin-sectioning TEM on four species of Spiroplasma, in vitro and/or in vivo, indicated that their helices commonly possess one tapered end (tip structure) and one blunt or round end. These tip structures appeared morphologically different from the rest of the helix, exhibiting an electron-dense conical or rod-shaped core. In thin sections of the midgut of the leafhopper Dalbulus elimatus, the tip structures of Spiroplasma kunkelii in the midgut lumen were mostly aligned between microvilli, perpendicular to the apical plasma membrane of epithelial cells. These tip structures appeared frequently attached or closely apposed to the plasma membrane, in which cup-shaped invaginations close to the tips were observed. Pleomorphic forms of spiroplasma, enclosed in membranous vesicles, were found in the cytoplasm of the midgut epithelial cells. These findings suggest that the tip structure may be involved in the orientation and attachment of spiroplasma helices in relation to their host cells, and thus may be functionally comparable to the attachment organelle of mycoplasmas. Additionally, pili-like structures were observed by negative-staining TEM on the surface of Spiroplasma melliferum, and in thin sections of S. kunkelii infecting the leafhopper vector Dalbulus gelbus. Abbreviations CSS Corn stunt spiroplasma - SEM Scanning electron microscopy - TBS Tris-buffered saline - TEM Transmission electron microscopy     

A maize defective-kernel mutant (longcell) characterized by tubular cells, severe morphological alterations and induction of cell death

Seeds of the longcell mutant in maize (Zea mays L) have a defective-kernel phenotype: the embryo aborts at the early coleoptilar stage and the endosperm is reduced in size. Mutant embryos have severe alterations in morphogenesis. They have a suspensor-, an embryo axis- and a scutellum-like structure, but the shoot apical meristem (SAM) is not formed. Scanning electron microscopy showed that most of the cells in longcell embryos are tubular and abnormally enlarged. The level of expression of several genes involved in basic metabolism is not severely affected during early and mid embryogenesis, but storage molecule accumulation is reduced. Genes which in normal conditions are only expressed after germination, are expressed during kernel development in the longcell seeds. Mutant embryos undergo cell death in late embryogenesis. Nuclei in dying embryos are TUNEL positive, and different genes coding for hydrolytic enzymes are up-regulated. The expression of genes related to oxidative stress is also altered in longcell embryos. These results lead us to suggest that the longcell mutant may be cytokinesis-defective.     

Gibberellin Effect on Tryptophan Metabolism, Auxin Destruction, and Abscission in Coleus

Application of gibberellic acid (GA) to the apical region of the stem enhances ¹⁴CO₂ release from tryptophan-l-¹⁴C in cell free preparations of the apical region. Although GA when applied to the apical region markedly accelerates abscission rates of debladed petioles at the 4th node, the enhancement effect on tryptophan metabolism appears to be restricted to the apical bud region. The increased levels of diffusible auxin in Coleus stems, observed earlier by Muir and Valdovinos (1965), appear to be due to the GA effect on auxin precursor conversion rather than to an altered rate of auxin destruction. GA pre-treatment does not significantly alter destruction rates of auxin in the stem tissue. This is demonstrated by the release of ¹⁴CO₂ from IAA-1-¹⁴C by sections of internode tissue. While a multiple deblading pattern retards abscission of debladed petioles considerably, application of GA to debladed petioles at the basal region of the stem restores the normal rates of abscission at debladed distal nodes. No significant change in the abscission rates at treated nodes is observed. The GA effect on abscission at distal nodes is attributed to the effect of the growth substance on auxin precursor conversion in the apical region. In these experiments, as in the case of plants treated in the apical region with GA, auxin destruction rates in the stem are not altered significantly.     

Effect of Gibberellic Acid and Cycocel on Tryptophan Metabolism and Auxin Destruction in the Sunflower Seedling

A comparative study of tryptophan conversion in different regions of the sunflower seedling indicates that the regions most active in converting tryptophan on a pathway to auxin are the root apical segments and young leaves; next highest in activity is the cotyledonary tissue. The stem apex proper with leaf primordia is less active than the above regions in converting the auxin precursor. Hypocotyl tissue was observed to be least active. Pre-treatment of the apical bud region of the stem with gibberellic acid (GA) gives rise to tryptophan conversion rates which are 2.1 times those in untreated seedlings. The enhanced tryptophan conversion in the apical bud is followed by an increased elongation rate of the 1st internode which is 2.2 times that in the 1st internode of untreated seedlings. Treatment of the seedlings with Cycocel [(2-chloroethyl)trimethylamnionium chloride] does not reduce tryptophan conversion in the apical bud region of the seedling although elongation of the stem is greatly retarded. Indoleacetic acid (IAA) destruction in cell free preparations as well as in whole sections of the elongating region of the seedling stem was studied. IAA-1-¹⁴C destruction rates with the release of ¹⁴CO₂ in whole sections of 1st internode tissue were approximately 3 times those in cell free preparations of the same region. No significant changes in IAA destruction rates in seedlings pre-treated with GA or Cycocel were observed.     

The transport of radiolabeled indoleacetic acid and its conjugates in nodal stem segments of Phaseolus vulgaris L.

The transport of radiolabeled indoleacetic acid (IAA), and some of its conjugates, was investigated in nodal stem segments of Phaseolus vulgaris L. Donor agar blocks containing either [2-acetyl-¹⁴C]-IAA; [2-acetyl-¹⁴C]-indole-3-acetyl-L-aspartate (IAAsp); [2-acetyl-¹⁴C]-indole-3-acetyl-L-glycine (IAGly); or [2-acetyl-¹⁴C]-indole-3-acetyl-L-alanine (IAAla) were placed on either the apical or basal cut surface of stem segments each bearing an axillary bud at the midline. In some experiments, a receiver block was placed on the end opposite to the donor. After transport was terminated, the segments were divided into five equal sections plus the bud, and the radioactivity of donors, receivers and each part of the stem segment was counted.For all four substances tested, the amount of ¹⁴C transported to the axillary bud from the base was the same or greater than that from the apical end. After basipetal transport, the distribution of ¹⁴C in the segment declined sharply from apex to base. The inverse was true for acropetal transport. Transport for the three IAA conjugates did not differ substantially from each other.The IAA transport inhibitor, N-1-naphthylphthalamic acid (NPA), inhibited basipetal ¹⁴C-IAA transport to the base of the stem segment but did not alter substantially the amount of ¹⁴C-IAA recovered from the bud. Transport of ¹⁴C-IAA from the apical end to all parts of the stem segment declined when the base of the section was treated with nonradioactive IAA. Taken together with data presented in the accompanying article [Tamas et al. (1989) Plant Growth Regul 8: 165–183], these results suggest that the transport of IAA plays a role in axillary bud growth regulation, but its effect does not depend on the accumulation of IAA in the axillary bud itself.