高等植物中原生质细胞间运动研究的进展

20世纪初已在植物营养组织和生殖体中观察到细胞核或染色质在细胞间的穿壁迁移.但多年来涉及这一研究的学者大多倾向于将它看做是人为赝象而未予深究.50年代中期,自吴素萱在葱(Allium L.)、蒜(Allium sativum L.)等植物中重新发现了核穿壁运动现象以来,我国植物细胞与生理学家随之进行了系统与广泛的探讨.穿壁运动的研究及进展主要包括两个方面.一方面是营养组织中原生质的细胞间运动,由吴素萱、娄成后合作主持的有关研究项目着重与营养组织中有机物的运输、分配与再利用生理过程相联系.多年来积累的成果表明:1)核物质的穿壁运动是广泛存在于植物营养组织中的固有正常现象,是一定生理状态下有机物在细胞间运输与细胞内含物再分配的一种方式;2)通过对蒜芽鞘、小麦(Triticum aestivum L.)珠心、胚乳等组织的显微活体观察与亚显微结构分析,揭示与记录了原生质在细胞间运动的全过程与动态细节,发现参与穿壁过程的不限于核,核和胞质各组分可同时、亦可分别借自身的主动伸缩穿壁迁移;呈现穿壁运动的组织中,壁上部分胞间连丝结构剧烈变更,胞间通道明显扩大,可达100～300 nm,核或胞质可经此开放通道迁移而不致有损通道外沿质膜的完整;3)原生质穿壁运动的动力来自细胞的能量代谢,与微丝活动密切有关,穿壁的原生质组分呈现活跃的ATP酶活性.近期的研究表明,小麦胚乳细胞中作活跃伸缩运动的胞质纤索是由F肌动蛋白集束组成,肌动蛋白纤丝可跨胞分布而存在于常态胞间连丝中,从而使相邻细胞的胞质骨架得以实现胞间连续.另一方面是生殖体中染色质的穿壁运动研究,主要在郑国主持下开展,着重探讨花粉母细胞间染色质穿壁现象的普遍性及其与遗传、变异和进化的关系;对多种植物的系统观察证明,染色质穿壁出现在减数分裂前期Ⅰ的凝线期,染色质通过胞质通道在一系列细胞间依次迁移;由电镜观察可见,参与穿壁运动的还包括胞质的多种成员;穿壁运动的动力被认为由原生质的收缩蛋白提供.郑国等的研究成果表明,染色质穿壁运动可导致染色体数目、结构的改变,由此产生的部分有活力的配子体经受精后有可能导致新一代个体出现多倍体或非整倍体,引起遗传性状的变异.     

The Specificity of the Feulgen Reaction for Thymonucleic Acid

The results of experiments on the specificity of the Feulgen reaction for thymonucleic acid do not substantiate the observations of Carr. The staining is not localized in the nucleus because of the destruction of cytoplasmic constituents following acid hydrolysis or because of the absorbing power of chromatin, since the cytoplasm and nucleolus can still be stained by numerous dyes. The effects of factors such as the acid hydrolysis and sulfurous acid washing baths upon the cytologic distribution of dye were studied on tissues stained with (1) fuchsin-sulfurous-acid (Feulgen) reagent, (2) fuchsin-sulfurous-acid reagent colorized by the addition of formaldehyde, (3) basic fuchsin in one-tenth normal HCl, and (4) basic fuchsin in distilled water. Under comparable conditions, important differences between these stains were found in the effects of preliminary hydrolysis; rapidity of staining and destaining; extractability of dye from tissues by water, alcohol, and sulfurous acid solution; rate of fading from exposure to light; localization of stain in tissues; and differences in hue. After treating tissues with desoxyribonuclease, an enzyme which acts only upon thymonucleic acid, cells do not stain with the Feulgen technic. Following removal of nucleic acid from chromatin by hydrolysis, attempts to demonstrate an absorption of thymonucleic acid upon the residual nuclear protein were unsuccessful.

The evidence for and against the specificity is discussed. In agreement with most other investigators, on the basis of the evidence in the literature as well as these experiments, it is concluded that when properly controlled the Feulgen reaction is relatively specific for thymonucleic acid.     

Pervasive migration of organellar DNA to the nucleus in plants

A surprisingly large number of plant nuclear DNA sequences inferred to be remnants of chloroplast and mitochondrial DNA migration events were detected through computer-assisted database searches. Nineteen independent organellar DNA insertions, with a median size of 117 by (range of 38 to >785 bp), occur in the proximity of 15 nuclear genes. One fragment appears to have been passed through a RNA intermediate, based on the presence of an edited version of the mitochondrial gene in the nucleus. Tandemly arranged fragments from disparate regions of organellar genomes and from different organellar genomes indicate that the fragments joined together from an intracellular pool of RNA and/or DNA before they integrated into the nuclear genome. Comparisons of integrated sequences to genes lacking the insertions, as well as the occurrence of coligated fragments, support a model of random integration by end joining. All transferred sequences were found in noncoding regions, but the positioning of organellar-derived DNA in introns, as well as regions 5 and 3 to nuclear genes, suggests that the random integration of organellar DNA has the potential to influence gene expression patterns. A semiquantitative estimate was performed on the amount of organellar DNA being transferred and assimilated into the nucleus. Based on this database survey, we estimate that 3–7% of the plant nuclear genomic sequence files contain organellar-derived DNA. The timing and the magnitude of genetic flux to the nuclear genome suggest that random integration is a substantial and ongoing process for creating sequence variation.Correspondence to: J.L. Blanchard     

A simple polychrome stain for conventionally fixed Epon-embedded tissues.

The described technique, based upon a one-step Mallory-Heidenhain stain, can be applied as a routine stain for glutaraldehyde or OsO4 fixed, Epon embedded tissues of various organs. The technique consists of a short treatment of the sections with H2O2, a nuclear staining with celestine blue B and a final staining in a modified Cason's solution. The different tissue and cell components are displayed as follows: dark brown nuclei, yellow cytoplasm, red collagen fibers and blue elastic fibers. Intracytoplasmic components as glycogen and mucus are stained respectively blue and violet, whereas other inclusions such as leucocyte granules are colored orange to red.     

Flow cytometric analysis of polysomaty and in vitro genetic instability in potato

The analysis of nuclear DNA contents in various tissues of potato genotypes showed that flow cytometry is a rapid method to characterize large populations of cells for polysomaty, that is, the occurrence of cells with normal DNA levels together with cells containing endoreduplicated nuclei. The proportion of endoreduplicated nuclei varied in different tissues and genotypes of potato. The analysis of callus and cell cultures showed that the temporal changes in nuclear DNA contents during in vitro growth can be followed and the degree of polyploidization quantified. It is concluded that flow cytometry is a highly suitable method to detect ploidy changes in differentiated plant tissues and calli which are often not amenable for chromosome number determination.     

A Simple Polychrome Stain for Conventionally Fixed Epon-Embedded Tissues

The described technique, based upon a one-step Mallory-Heidenhain stain, can be applied as a routine stain for glutaraldehyde or OsO₄ fixed, Epon embedded tissues of various organs. The technique consists of a short treatment of the sections with H₂O₂, a nuclear staining with celestine blue B and a final staining in a modified Cason's solution. The different tissue and cell components are displayed as follows: dark brown nuclei, yellow cytoplasm, red collagen fibers and blue elastic' fibers. Intra cytoplasmic components as glycogen and mucus are stained respectively blue and violet, whereas other inclusions such as leucocyte granules are colored orange to red.     

Newer applications of the histological stain prepared from Pterocarpus santalinus

A histological stain prepared from the heartwood of Pterocarpus santalinus Linn. has been found to be an excellent nuclear stain for various cells of animal and plant origin. As an elastic tissue stain, the results are comparable to standard elastic tissue stains. The striations of voluntary muscle fibers are well shown. The Nissl granules and fibers of cranial nerves in the pons are visualized. When counterstained with light green, it differentially stains muscle and fibrous tissue. The stain can be used as counterstain with certain histochemical procedures with satisfactory results. The preparation and use of this versatile stain are described.     

The plant cell nucleus is constantly alert and highly sensitive to repetitive local mechanical stimulations

When mechanical stimulation is applied to a plant cell, the nucleus usually shows oriented movement to the site of stimulation (as a defensive response). Former researchers have revealed that applying mechanical pressure to plant tissues could line up cell division plane. A proposal, therefore, was put forward that cells inside plant tissue could receive mechanical signals from their growing neighbors to adjust their nuclear position and thus regulate the orientation of their dividing plane in order to form characteristic morphology of plant organs. To explore nuclear capacity and sensitivity to rapidly changing signals, multiple mechanical stimulations were applied to the same plant cell at intervals, either locally or at distance. The results revealed that the nucleus was highly sensitive to mechanical stimulations. It responded quickly to both local and distant stimulation by showing oriented movement toward the stimulation site. The nucleus was able to respond immediately to a second stimulation (no time lag) by starting up a second oriented movement toward the new signal; the completion of nuclear oriented movement to a first site of stimulation was not necessary for startup of a subsequent movement track to a second stimulation site, regardless of whether the second stimulation was applied ahead of or behind the moving nucleus. The nucleus responded to a second stimulation without loss of velocity, whether or not it was in a resting or moving state. This novel finding favors the proposal that growing tissues adjust the location of nuclei in cells by varying mechanical pressures; they thus control cell division according to a plan whereby organs and their constituent tissues develop in an orderly, specified manner. It appears that the enhanced sensitivity of plant cells to mechanical pressure is necessary not only in response to the external environment, but also to the developmental microenvironment inside the tissues.     

Newer Applications of the Histological Stain Prepared from Pterocarpus Santalinus

A histological stain prepared from the heartwood of Pterocarpus santalinus Linn, has been found to be an excellent nuclear stain for various cells of animal and plant origin. As an elastic tissue stain, the results are comparable to standard elastic tissue stains. The striations of voluntary muscle fibers are well shown. The Nissl granules and fibers of cranial nerves in the pons are visualized. When counterstained with light green, it differentially stains muscle and fibrous tissue. The stain can be used as counterstain with certain histochemical procedures with satisfactory results. The preparation and use of this versatile stain are described.     

Crystal Violet as A Nuclear Stain for Gaffkya Tetragena and Other Bacteria

A method is given to stain the nucleus of Gaffkya tetragena and other bacteria. Only water solutions of crystal violet, mercuric chloride, and nigrosin are used. While the application of heat (50°C. for 20 seconds) is not absolutely necessary, it facilitates the decolorizing process. Some of the nuclei of cells from old cultures were found in various stages of division. The nucleus of a mature cell divides, and each daughter nucleus undergoes a second division usually in a plane at right angles to that of the first division. The nuclear division indicates cell development in units of four cells each. The method was found reliable for demonstrating the nucleus at various periods and stages of growth.     

Involvement of contractile proteins in the changes in consistency of oocyte nucleoplasm of the newt Pleurodeles waltlii

In the present work, we show that actin is present in considerable quantities in the oocyte nucleus of the newt Pleurodeles waltlii. The nuclear sap, extracted in saline buffer containing Ca++, is fluid. DNAase I inhibition assays have shown that 90% of actin is under a globular state in such conditions. Chelation of Ca++ by EGTA leads to the formation of a nuclear gel composed of individual microfilaments. This nuclear gel contains approximately 50% of total nuclear actin in a filamentous form. Phalloidin, a drug known to stabilize F-actin, induces the formation of a network of actin cables in the nuclei. This network contains nearly 100% of total nuclear actin in the filamentous form. The observation of the cables in the electron microscope shows that they are made of tightly associated microfilaments to which RNP- like particles are bound. The actin antibodies stain the cables and the particles by the indirect immunoperoxidase technique; myosin antibodies mainly stain the particles. The formation of the phalloidin-induced network seems to require the presence of Ca++, Mg++, and ATP. We propose a scheme for the regulation of the supramolecular forms of actin in oocyte nuclei in which a delicate equilibrium seems to exist between globular actin, microfilaments, and actin cables. This equilibrium would be controlled by the concentration of Ca++, ATP, and various actin-associated proteins.     

Gallocyanin as A Nuclear Stain

Gallocyanin has been used successfully as a nuclear stain. Sections are cut by the freezing method of either fixed or unfixed tissue. The tissues are warmed (not exceeding 70°C.) for 2-4 minutes in the gallocyanin solution. A counterstain may be used if desired. The most effective are Biebrich scarlet, phloxine, or eosin Y. The sections are then dehydrated and mounted in clarite. The nuclear pattern is clearly demonstrated and the sections are permanent.     

A re-examination of the role of the nucleus in generating the circadian rhythm in Acetabularia.

The role of the nucleus in the generation of the circadian rhythm in Acetabularia has been nuclear. Early experiments showed that the plant could exhibit a circadian rhythm in the absence of a nucleus. However, other experiments appeared to show that the nucleus could impart phase information to the rhythm, and so therefore must be a part of the system that generates the rhythm. We have conducted experiments similar to these--in particular, one in which the nuclear end of the plant was entrained on a light-dark cycle that was opposite that of the rest of the plant. The phase of the free-running rhythm of this type of plant is not consistent with the conclusion that the nucleus is part of the circadian oscillator. We have also tried entraining opposite ends of plants with no nuclei on opposite light-dark cycles. The ultimate phases of these plants appear to be nearly random. A possible interpretation of these experiments is discussed.     

Brilliant Cresyl Blue as a Stain for Plant Chromosomes

Methods are proposed for staining plant chromosomes with the dye brilliant cresyl blue, and for making these stained preparations permanent by using polyvinyl alcohol mounting medium.

The stain, which is composed of 2% brilliant cresyl blue in 45% aqueous acetic or propionic acid, is used with fixed material in making smear preparations. The technics for staining are similar to those employed in the aceto-carmine method.

The mounting medium is made by mixing 56% polyvinyl alcohol, which is diluted in water to the consistency of thick molasses, with 22% lactic acid and 22% phenol by volume. The permanent slides are made by floating off the cover slip of the temporary slide in 70% alcohol, then applying the mounting medium and replacing the cover slip.

The chief advantages of the methods described are:

1)The preparation of the stain is rapid and simple. The batch of stain will be good with the first try.

2)The staining procedure in some instances is shorter than when using aceto-carmine.

3)The stain shows a high degree of specificity for nuclear structures and gives better results than aceto-carmine when used on certain plant tissues.

4)A minimum number of cells is lost in making the slides permanent when using polyvinyl alcohol mounting medium as the slide and cover slip are run through only one solution prior to mounting.

5)The mounting medium dries rapidly and this shortens the time required before critical examination of the permanent mounts can be made.     

Autonomous regulation of free Ca2+ concentrations in isolated plant cell nuclei: a mathematical analysis

Experiments performed on nuclei isolated from animal or plant cells have provided evidence that the nucleus generates directly specific nucleoplasmic calcium transients in response to external stimuli. Recent data suggest that isolated plant nuclei might be considered as a closed system where the nuclear concentration of free calcium would be regulated by reversible movements between the nucleoplasm and nuclear stores. We have addressed the relevance of this hypothesis by developing a mathematical approach to simulate nucleoplasmic calcium dynamics generated under various pH and temperature conditions. Here, we show that the experimental results could be explained provided that calcium channels as well as systems transporting calcium are present on the inner nuclear membrane. The putative channels would allow the entry of calcium into the nucleoplasm whereas the elusive transporting system(s) would contribute to replenish the nuclear stores. The simple proposed model is versatile enough to explain and predict autonomous changes in free calcium in the nucleoplasm of isolated plant nuclei.     

Staining with chromoxane cyanine R.

Since Pearse in 1957 introduced chromoxane cyanine R as a dual nuclear and cytoplasmic stain there have appeared numerous procedures for use of this dye. These have differed widely, sharing in common mainly the implication that each is best. A defendable procedure has been developed on an experimental basis and is reported here. Four stock solutions are needed: (1) a 0.2% solution of chromoxane cyanine R in 0.5% aqueous H2SO4 (v/v); boil this solution for 5 min, (2) 10% FeCl3 in 3% HCl, (3) 1% aqueous HN4OH, and (4) 1% HCl in 70% ethanol. The staining solution: 40 ml of dye solution, 2 ml of FeCl3 solution, 8 ml H2O. Dewax and hydrate sections and stain for 10 min. If a myelin sheath stain is desired differentiate for 1 min in solution (3). For a nuclear stain differentiate for 1 min in solution (4). The nuclear stain when counterstained with eosin closely resembles the routine hematoxylin and eosin. Histochemical tests show that the functional group for myelin staining contains nitrogen, and probably hydrogen bonding is involved. The nuclear stain involves a different functional group and possibly neither electrostatic nor hydrogen bonding.     

Visualizing eggs of Nesidiocoris tenuis (Heteroptera: Miridae) embedded in tomato plant tissues

Nesidiocoris tenuis (Heteroptera: Miridae) is a predator of some major pests of Solanaceae crops, yet it is scarcely used in biological control because it also feeds on plants and may damage crops. The study of N. tenuis biology may promote the ability to use it as a biological control agent. Because N. tenuis, like some other insect taxa, oviposits into plant tissues, its eggs are hard to detect. This limits our ability to study N. tenuis – plant interactions. We therefore looked for a staining method for plant‐embedded eggs, which will allow their detection within tomato plants, a common host of N. tenuis. We first used lactophenol solution with acid fuchsin to stain eggs inside tomato foliage. Because of the high toxicity of lactophenol, we later substituted lactophenol with a lactoglycerol solution, which was found to be similarly efficient. Five minutes immersion in the staining solution at 80°C followed by a two‐minute soak in hot water made the eggs stain deep red, while the foliage became transparent and was stained weak red. Eggs within leaves were easily visible under 10–30× magnification with sub‐stage lighting; top‐lighting was needed for the detection of eggs embedded in less‐transparent tissues such as stems. This rapid staining method improves the ability to study some important biological aspects of N. tenuis, such as its fecundity. Also, the elimination of phenol made the method cheaper and safer to use. Finally, this method may be adapted for other arthropod–plant systems.     

Staining with Chromoxane Cyanine R

Since Pearse in 1957 introduced chromoxane cyanine R as a dual nuclear and cytoplasmic stain there have appeared numerous procedures for use of this dye. These have differed widely, sharing in common mainly the implication that each is best. A defendable procedure has been developed on an experimental basis and is reported here. Four stock solutions are needs. (1) a 0.2% solution of chromoxane cyanine R in 0.5% aqueous H₂SO₄ (v/v); boil this solution for 5 min, (2) 10% FeCl₃ in 3% HCI, (3) 1% aqueous NH₄OH, and (4) 1% HCI in 70% ethanol. The staining solution: 40 ml of dye solution, 2 ml of FeCl₃ solution, 8 ml H₂O. Dewax and hydrate sections and stain for 10 min. If a myelin sheath stain is desired differentiate for 1 min in solution (3). For a nuclear stain differentiate for 1 min in solution (4). The nuclear stain when counterstained with eosin closely resembles the routine hematoxylin and win. Histochemical tee show that the functional pup for myelin staining contains nitrogen, and probably hydrogen bonding is involved. The nuclear stain involves a different functional group and possibly neither electrostatic nor hydrogen bonding.     

The structure of the endoplasmic reticulum revealed by osmium tetroxide-potassium ferricyanide staining

Postfixation of plant tissues with a mixture of osmium tetroxide and potassium ferricyanide (OsFeCN) yields a selective staining of the endoplasmic reticulum (ER) and nuclear envelope (NE). The other cytoplasmic organelles and inclusions are evident, but by comparison with the NE-ER they are weakly contrasted. Demarcation of the NE-ER results from the enhanced deposition of an electron-opaque reaction product on the inner leaflet of the membrane that extends into the cisternal space. The procedure thus renders the NE-ER readily apparent even when the elements are sectioned parallel to their surface and makes it possible to easily visualize their cellular pattern. Ultrastructural studies reveal with clarity tubular reticula and fenestrated lamellae that are extensively interconnected into one continuous membrane system. Problems with the OsFeCN procedure include the inability of the reagent to stain the NE-ER in all cells of a tissue, the occasional staining of non-ER such as dictyosomal cisternae and plastids, and the failure to selectively stain the NE-ER in protoplasts or single wall-less cells. Results obtained with OsFeCN are compared with other ER fixatives and stains including potassium permanganate and zinc iodide-osmium tetroxide. Despite its problems, under optimal circumstances OsFeCN is judged to be superior to other stain-fixatives for selectively contrasting the NE-ER compartment and is recommended generally for ultrastructural investigations.