Staining and osmotic properties of young gametophytes ofPolypodium vulgare L. and their bearing on rhizoid function

Summary The rhizoids of gametophytes ofPolypodium vulgare L. rapidly absorb vital stains whereas the protonemal cells are impermeable to these stains, which can only enter the cells from the rhizoids. The protonemal cells which bear rhizoids were found to have a slightly higher osmotic equivalent than did the rhizoids or the protonemal cells on either side. From the results of several staining procedures it was demonstrated that the rhizoid walls contain free carboxyl groups and thus possess cation exchange properties. Most of the carboxyl groups are probably present in a yellow-brown wall matrix substance, which shows high resistance to acid and alkali extraction. The precise nature of this substance has not been determined but it could be an acid mucopolysaccharide. Carboxyl groups are detectable in the protonemal cell walls only after saponification and are probably esterified in the untreated wall. Several other chemical and physiological differences were found between the rhizoids and the protonemal cells and it was concluded that the specific properties of the rhizoids are related to their function as organs of uptake.     

An Interpretation of Several Cell Wall Stains Applied to Heat-Fixed Bacillus subtilis On Glass Slides

The Dyer, Chance, alcian blue, and tannic acid-crystal violet wall stains for bacteria were compared. All gave apparent cell wall widths which were considerably greater than those demonstrated by electron microscope methods. The exaggerated width, as seen by the light microscope, was apparently due to staining of a portion of the cytoplasmic material adjacent to the wall, and in addition, to a precipitate on the surface of the cell produced by the Dyer and the Chance stains. Heat-fixed cells on slides were shown to retain considerable height or third dimension, being about half as high as wide. Moreover, such cells had their walls flattened against the slide to form a collar-like projection around the periphery of the shrunken protoplasm. This latter effect alone was not sufficient to explain the exaggerated wall thickness shown by staining. For obtaining reliable measurements of cell widths, the nigrosin negative stain was found to be as good as any, provided that the thickness of its film were controlled. Negative stains were used so that comparisons of cell widths shown by them and by positive stains could be made. This, in turn, facilitated the detection of the apparent widening of cell bodies caused by dye precipitates on their surfaces.     

Cytochemical localization of calcium in cap cells of primary roots of Zea mays L

The distribution of calcium (Ca) in caps of vertically- and horizontally-oriented roots of Zea mays was monitored to determine its possible role in root graviresponsiveness. A modification of the antimonate precipitation procedure was used to localize Ca in situ. In vertically-oriented roots, the presumed graviperceptive (i.e., columella) cells were characterized by minimal and symmetric staining of the plasmalemma and mitochondria. No precipitate was present in plasmodesmata or cell walls. Within 5 min after horizontal reorientation, staining was associated with the portion of the cell wall adjacent to the distal end of the cell. This asymmetric staining persisted throughout the onset of gravicurvature. No staining of lateral cell walls of columella cells was observed at any stage of gravicurvature, suggesting that a lateral flow of Ca through the columella tissue of horizontally-oriented roots does not occur. The outermost peripheral cells of roots oriented horizontally and vertically secrete Ca through plasmodesmata-like structures in their cell walls. These results are discussed relative to proposed roles of root-cap Ca in root gravicurvature.     

Isolation and Identification of Native Auxins in Marine Algae

Endosperm cell walls were isolated from rice grains and their chemical composition was analyzed. The cell walls were composed of cellulose microfibrils and matrix phase which consisted of hemicellulose and pectic substances. Hemicellulose mainly comprised arabinoxylan, accompanied by a small amount of glucose-containing polysaccharide. Pectic substances contained polygalacturonides, some of which had side chains containing neutral sugars such as galactose and arabinose. Amino acid analysis of these fractions suggested that hydroxyproline-containing glycoproteins were contained in these cell walls and firmly bound to cellulose microfibrils.     

The eye of the soldier beetle Chauliognathus pulchellus (Cantharidae).

The soldier beetle eye is unusual in having large optically isotropic corneal cones which project inwards from a thick isotropic cornea. Refraction is mainly at the corneal surface. Calculation shows that the first focal plane is near the tip of the cone, from which the optical pathway continues as a crystalline tract. At the distal end of the crystalline tract, 3 micrometer in diameter, the four cone cells enclose the proximal tip of the corneal cone; at the proximal end they enclose the distal tip of a long fused rhabdom rod. The eye is remarkable in that there are two classes of retinula cells; four cells contribute to the long thin axial rhabdom, 2 micrometer in diameter and 120 micrometer long, and the other four cells form two rounded rhabdoms, 10 x 4 micrometer in cross-section and 20 micrometer deep, which lie to one side of the optical axis. The physiological properties of individual retinula cells were measured by intracellular recording. The retinula cells are of three spectral types with peaks near 360, 450 and 520--530 nm. Except by the criterion of spectral sensitivity, the retinula cells sampled could not be sorted into more than one class. The measured value of the acceptance angle, near 3 degrees in the dark-adapted state, is consistent with the hypothesis that all sampled cells were of the anatomical type that participate in the central rhabdom rod. A calculation of the theoretical field size of individual retinula cells from measurments of refractive index and lens dimensions predicts that cells which participate in the central rhabdom will have acceptance angles near 3 degrees. The conclusion, therefore, is that only one anatomical type of cell has so far been sampled.     

Changes in Morphometry and Elemental Composition of Robinia pseudoacacia Pulvinar Motor Cells During Leaflet Movements

Changes in shape and size of Robinia pulvinar cortical cellsin relation to leaflet movements have been investigated usingan image processing system applied to drawings of transverseand longitudinal pulvinar sections. Both the size and shapeof cell sections underwent change during movement. The dorsal-leftside region of the cortex has been characterized as the extensorregion which increases turgor during opening. Morphometric changesoccur throughout the cortical motor cells except in the threeor four inner layers. K, Cl, S, and Ca distribution in cellwalls and protoplasts of inner and outer motor cells have beenmeasured with X-ray microanalysis. The distribution patternof K and Cl shows that these ions are mainly responsible forturgor changes. K and Cl were simultaneously depleted in apoplastand protoplast, which suggests that cell walls do not possessa high enough ionic reservoir during Robinia leaflet movements.Ca was always higher in flexor cell walls than in extensor regionsof closed pulvini. This fact could be related to a lower abilityto extend of flexor cells which underwent fewer morphomerticchangrs during movement.     

An electron microscopic study of the location of teichoic acid and its contribution to staining reactions in walls of Streptococcus faecalis 8191.

The location of the glucosylated teichoic acid in whole cells and isolated walls of Streptococcus faecalis 8191 has been investigated using ruthenium red, gold-labelled concanavalin A and concanavalin A-peroxidase-diaminobenzidine. Dense laminae were revealed in sections of osmium-fixed walls stained with ruthenium red which corresponded to similar regions stained by uranyl and lead. Such regions were not seen after teichoic acid had been extracted, suggesting that the uptake of stain was by teichoic acid. However, these regions were not labelled on exposure to gold concanavalin A or concanavalin A-peroxidase-diaminobenzidine; these stains indicated that teichoic acid was situated between the dense laminae, although the distribution of stain could have been due to the inability of the concanavalin A stains to penetrate deeply. Chemical binding studies showed that the teichoic acid was the major uranyl binding component in isolated walls, from which it might be inferred that teichoic acid was located in the densely staining regions. However, since osmification significantly increased the binding of uranyl (and lead stains) to non-teichoic acid material, such an inference was not necessarily valid. It is concluded that the presence of teichoic acid can be demonstrated in certain regions of the wall by concanavalin A, but its presence in densely staining regions has not been established. These experiments therefore suggest that teichoic acid may not be intimately associated with the mechanisms that generate contrast patterns in stained sections of cell walls of Streptococcus faecalis.     

Contributions of the surface of the root tip to the growth of Zea roots in soil

The development of the epidermal layer of roots of Zea is traced from the quiescent centre to the zone where root hairs develop. In the zone of cell division a three layered coat forms on the outside of the epidermal cells consisting of the outer epidermal walls, overlaid by a two-layered pellicle composed of a thick fibrillar inner layer of polysaccharide, and a thin fibrillar outer layer of protein. The epidermal cells divide several times in the same longitudinal file but rarely across a radius to give a new longitudinal file. Thus, the radial walls become much thicker than all but the original transverse walls, and packets of up to 32 daughter cells derived from a single initial may be distinguished. The pellicle develops during these divisions as a continuum over the outer walls of the daughter cells. It is proposed that the pellicle provides a stiffening to the forward end of the root which permits it to penetrate soil without bending. Support for this hypothesis is shown by the Zea mays mutant Ageotropic in which the pellicle is absent, the epidermal surface is disorganized, and which grows crookedly through soil. In the zone of extension growth of normal roots of two Zea species the pellicle thins and disappears. Circumferential strips of the pellicle were peeled off the young epidermal cells and could be stretched to twice their length. This deformation is partly the result of the pellicle stretching and breaking above the attachments of the radial walls. After normal thinning of the pellicle, detachment of the radial walls at their outer ends produces a corrugated surface in the proximal zone of the root tips. In dicotyledons (e.g., soybean), there is no similar pellicle, but a stiff root tip is produced by a long multi-layered root cap, the proximal portion of which covers the elongating epidermal surface.     

Structure of the endosperm in the mature seed of Melilotus alba]

The results of the exam at the light, the fluorescence and the scanning electron microscope of the endosperm of Melilotus alba mature impermeable seeds are reported. Cryostat sections, semithin sections and squashes are observed. Melilotus alba endosperm is variable in thickness and envelopes cotyledons and radicle. Its "aleurone" layer is one-cell thick, while the number of layers of its internal cells varies in relation to the location in the seed. In the aleurone cells, the cytoplasm and the outer portion of the wall are autofluorescent; tannic acid-ferric chloride stains the outer portion of the wall and allows to see clearly the inner thickenings, DAPI and haematoxylin demonstrate the presence of the nucleus. The cytoplasm of these cells is coloured by Sudan black b, and its fluorescence is enhanced by auramine and calcofluor white. Calcofluor white enhances the fluorescence of the outer portion of these walls, too, but is without effect on the non-autofluorescent thickening, indicating presence of cellulose only in the first case. Callose is absent. Also the thin autofluorescent walls of the endosperm inner cells react positively to calcofluor. These cells are very large, almost completely filled with "gelatinous" substances--the galactomannans--and very rarely contain a nucleus.     

Light microscopic detection of PAS-positive substances with thiosemicarbazide in freeze-substituted ovaries of Paspalum longifolium before pollination

Summary Localization of polysaccharides in the freeze-substituted, Eponembedded ovaries of Paspalum longifolium prior to pollination was carried out by periodic acid-Schiff's (PAS), periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-AgPr) and periodic acid-thiosemicarbazide-osmium (PA-TSC-OsO₄) reactions. The specificities of these three reactions were also studied. These three reactions are all effective for light microscopic demonstration of polysaccharides in the filiform apparatus, starch grains in the cells and PAS substance in the micropylar region. Nonspecific staining of the nucleoli of the egg and polar nuclei was observed in the PAS reaction. The PA-TSC-AgPr reaction is very specific for polysaccharides but its overall reaction takes a much longer period of time than the PAS reaction. The PA-TSC-OsO₄ reaction colors the cytoplasm and nuclei of most cells and therefore stains of the cell walls, especially those of the egg cell and synergids, do not stand out clearly. The synergid cytoplasm contains some amorphous polysaccharides and thus it colors even in PAS and PA-TSC-AgPr preparations. In the mature embryo sac, the egg and central cell as well as antipodals are vacuolated but the two synergids have no visible vacuoles under light microscope. Each synergid has a prominent filiform apparatus at the micropylar end, which stains intensely in all three preparations. The walls of the central cell and antipodals adjacent to the nucellar cells have many inward papillae which are also intensely stained in all three preparations. Starch grains are abundant in the ovary wall and usually absent in the nucellus and integuments. They are present in the egg, central cell and antipodals, but not in the two synergids.     

Fine structure of the mandibular gland in Japanese field vole (Microtus montebelli)

The mandibular glands of the Japanese field vole were examined by light microscopy, and transmission and scanning electron microscopies. The acinar cells contained light and coarse secretory granules, and reacted with PAS and stained slightly with AB; they were considered to be seromucous in nature. The acinar epithelium was composed of light and dark cells containing many secretory granules. The intercalated duct cells consisted of light cells possessing a few dense granules. A few cytoplasmic crystalloides of moderate density were observed in occasional light cells. The striated ducts were comprized of two distinct portions, a secretory portion and a typical striated portion without secretory granules. The epithelium secretory portion consisted of light and dark cells containing acidophilic granules and exhibited a sexual dimorphism in these granules: The male epithelia contained the granules of low to high densities, while the female epithelia had only dense granules being smaller than those in the males. The epithelium of typical striated portion was composed of light and dark cells containing fine vacuoles and vesicles.     

Rapid transbilayer movement of fluorescent phospholipid analogues in the plasma membrane of endocytosis-deficient yeast cells does not require the Drs2 protein.

Evidence is presented that endocytosis-deficient Saccharomyces cerevisiae end4 yeast cells rapidly internalize the fluorescent phospholipid analogues 1-palmitoyl-2-{6-[7-nitro-2,1, 3-benzoxadiazol-4-yl(NBD)amino] caproyl}phosphatidylcholine (P-C6-NBD-PtdCho) and P-C6-NBD-phosphatidylserine (P-C6-NBD-PtdSer). Both analogues redistributed between the exoplasmic and cytoplasmic leaflet with a half-time of < 15 min at 0 degrees C. The plateau of internalized analogues was about 70%. Transbilayer movement is probably protein-mediated, as the flip-flop of both analogues was very slow in liposomes composed of plasma-membrane lipids. Rapid analogue internalization was not abolished on depletion of intracellular ATP by about 90%. For P-C6-NBD-PtdCho only was a moderate decrease in the plateau of internalized analogues of about 20% observed, while that of P-C6-NBD-PtdSer was not affected. The Drs2 protein plays only a minor role, if any, in the rapid transbilayer movement of analogues in S. cerevisiae end4 cells. In S. cerevisiae end4 Deltadrs2 cells harbouring both an end4 allele and a drs2 null allele, about 60% and 50% of P-C6-NBD-PtdCho and P-C6-NBD-PtdSer, respectively, became internalized within 15 min at 0 degrees C. The preferential orientation of P-C6-NBD-PtdSer to the cytoplasmic leaflet is in qualitative agreement with the sequestering of endogenous phosphatidylserine to the cytoplasmic leaflet, as assessed by binding of annexin V. Virtually no binding of annexin V to spheroplasts of the parent wild-type strain or the mutant strains was observed. Likewise, no difference in the exposure of endogenous aminophospholipids to the exoplasmic leaflet between these strains was found by labelling with trinitrobenzenesulfonic acid. Thus, lipid asymmetry, at least of aminophospholipids, was preserved in S. cerevisiae end4 cells independently of the presence of the Drs2 protein.     

Flagellar adhesion ofCrithidia fasciculata to Millipore filters

Summary Millipore filters composed of mixed cellulose esters have been used to investigate the adhesion of flagella ofCrithidia fasciculata to a non-living substrate. After 4 days on the surface of a culture of the flagellate, one side of the filter was covered with a monolayer of cells. In most cases the flagella penetrated the pores of the filter and at one or more points along their length presumed sites of adhesion were marked by the presence of hemidesmosomes, characterized by a thickened inner leaflet of the flagellar membrane and the presence of large numbers of fine filaments. If the interstitial space of the filter was sufficiently large, the hemidesmosomes occurred at the apex of an evagination of the flagellar membrane. These evaginations are believed to arise by movement of the flagellum relative to the point of adhesion. The addition of distilled water causes de-adhesion of the flagellum and its withdrawal from the filter. The hypothesis that cells adhere to one another and to non-living substrates by different mechanisms is discussed in the light of the results obtained here.     

Cell division in yeasts : III. The biased, asymmetric location of the septum in the fission yeast cell, Schizosaccharomyces pombe

Living, dividing, log-phase fission yeast cells (178 pairs) were photographed by fluorescence microscopy of their fluorochromed walls. Analysis of the lengths, volumes, and fission scar distributions of these cells led to the following conclusions: the new septum is sited asymmetrically at division by length parameters, and the asymmetric site is biased toward the newer end (that end generated by the previous cell division) of the dividing cells. The volumes of the resultant sibs, however, are equal. Some rather simple models for siting of the septum are presumed untenable on the basis of the evidence.     

An electron microscopic study of the mature megagametophyte in Zea mays

With light microscopy maize megagametophytes stained with Alcian blue-periodic acid-Schiff (AB-PAS) reveal acid or neutral polysaccharides in various cell walls. Comparative fine structural studies were made of permanganate- or OsO₄-fixed material. Organelle distribution is random in the vacuolate and multinucleate antipodal cells; organelles are abundant; starch is scarce. Antipodal cell walls have large openings forming several syncytia. Some walls are papillate. In the central cell (primary endosperm cell) a thin peripheral layer of cytoplasm surrounds the large vacuole; organelle number is moderate; starch is abundant. The central cell wall is also papillate adjacent to the antipodals and around the egg apparatus. In the synergids organelle distribution is non-random; nuclei and numerous organelles occupy the micropylar cytoplasm of each synergid; vacuoles dominate the chalazal cytoplasm of these cells. The filiform apparatus stains with AB-PAS and is composed of both lightly and darkly stained amorphous material. In the egg, organelle distribution is perinuclear with vacuoles proximal to the micropyle; mitochondria are large, abundant and polymorphic; starch is abundant. Nucleolar diameter is five times greater in the central cell and egg than in the antipodal cells and ten times greater than in the synergids. Plasmodesmata occur in all cell walls within the gametophyte, but none appear in the gametophyte wall itself. It is suggested that the antipodals and synergids might be secretory, the latter probably being involved in pollen tube attraction, and that stored metabolites in the central cell and egg cytoplasm support rapid increase in metabolism following fertilization.     

Effect of static electric fields in root cells of Vicia faba (Fabaceae)

Serial electron microscopic sections were prepared from half-ripened meristematic root cells of Vicia faba (Fabaceae) which had been exposed gradually to 700, 1000, 2500, 3500, and 5000 V/m static electric fields during seven days with and without Zn and Cd electrodes. At the end of five weeks, wall loosenings and very small nuclei were observed in those root cells which were exposed to static electric currents from the lower side of the medium without electrodes, while abnormalities in cell formation, e.g., two cells with one nucleus, and GER occurrence were present in an electrolytic (Cd upward and Zn downward) medium. The cells exposed to a static current from the upper side of the medium had small nuclei and abnormal cell divisions in the electrolyte, but in a non-electrolyte very large nuclei and thicker cell walls were observed, the cytoplasm was dense with GER, pinocytosis was seen filled with mitochondria, and protoplast formation with big nuclei was seen in exocytosis.     

苏铁种子的种皮结构特征研究

对苏铁(Cycas revoluta Thunb.)种子的种皮进行了解剖研究,结果表明:苏铁种子的种皮分为外种皮、中种皮和内种皮3层结构.外种皮含有角质化的表皮细胞、薄壁细胞以及少量的厚壁细胞和异细胞,布有树脂道、气室和4束大维管束;中种皮主要由厚壁细胞群和木质化纤维组成,种孔端有一条缝合线,种脐端有3个孔;内种皮由多层干瘪的薄壁细胞和脉络状维管束组成,种孔端有一层椭圆状保护膜.对外种皮和内种皮维管束进行观察研究发现:外种皮和内种皮的维管束分布方式及其结构存在明显差异,外种皮的维管束由种脐端顺着种子弧形走向种孔端,内种皮的维管束呈脉络状,形成维管网贯穿其中;内、外种皮维管束中均存在多种不同样式的导管.     

Nuclear death in living Tetrahymena: the case of the haploid nuclei

During sexual conjugation in Tetrahymena the micronucleus divides meiotically, producing four haploid nuclei. While one of these nuclei divides mitotically to yield two genetically identical gametic pronuclei, a stationary pronucleus and a migratory pronucleus, the remaining three haploid nuclei degenerate and disappear. Typically, they migrate to the posterior end of the cell where they remain as residual bodies until they disappear. In the present study we asked whether degenerating haploid nuclei share any properties with apoptotic nuclei. Specifically, we wondered whether they would be stained by "apofluor", a combination of vital fluorescent indicators that differentially stains apoptotic nuclei in living cells. "Apofluor" includes acridine orange, which becomes trapped in acidic compartments and stains lysosomal bodies a brilliant orange-red, and Hoechst 33342, which binds to DNA and stains nuclei bright blue. With this dye combination, while ordinary nuclei stain blue, the apoptotic macronucleus stains first blue-green, then yellow, and finally orange. The progression in color is presumed to be due to the accumulation of protons in the apoptotic nucleus compartment. We found that three of the four post-meiotic haploid nuclei, those that are eliminated, were stained differentially green, then yellow, and then come to be indistinguishable from the orange lysosomal bodies. Differential staining can occur even while the nuclei are located at the anterior ends of the cells, and before the "viable" nucleus divides to form pronuclei. These results indicate that haploid nuclei in the process of degradation are differentially stained in living cells by "apofluor", and that the differential staining occurs early in the elimination process. Further, since the degenerating haploid nuclei are stained by "apofluor" it is likely that they are degraded by a mechanism similar to the elimination of the apoptotic macronucleus.     

A quantitative analysis of the interspecific plasmodesmata in the non-division walls of the plant chimera Laburnocytisus adamii (Poit.) Schneid.

Non-division walls in petals of the chimera Laburnocytisus adamii (Poit.) Schneid, were screened for the occurrence and distribution of symplasmic connections. The secondary plasmodesmata (PD) between epidermal cells of Cytisus purpureus Scop, and subepidermal cells of Laburnum anagyroides Medik. were compared with the PD of corresponding cell walls in petals of the two parental species. The non-division walls in the petals of L. adamii were traversed mainly by continuous PD and a few half-PD, both being grouped in pit fields. The secondary PD were characterized by a high percentage of branching (82%), with more than 40% consisting of a single strand at the Cytisus cell side interconnected by a median cavity with two strands of the Laburnum subepidermal cell. In addition, more than 30% of all PD showed secondary branching in the subepidermal wall portion. As a consequence, the cross-sectional areas of plasmodesmatal strands on each side of the central cavity differed remarkably in size, representing a “bottleneck” in the epidermal wall portion. In contrast, PD in the petals of the parental species were symmetrically branched. The comparison of cross-sectional areas of PD in the cell wall between the epidermis and subepidermis of petals of L. anagyroides showed a well-tuned system. The occurrence of half-PD in the intraspecific wall indicates a secondary origin. We conclude that, in the chimera, both genotypically different cells take part in the formation of the interspecific PD.     

SECONDARY PULVINUS OF ROBINIA PSEUDOACACIA (LEGUMINOSAE): STRUCTURAL AND ULTRASTRUCTURAL FEATURES

The structure of the secondary pulvinus of Robinia pseudoacacia has been examined together with ultrastructural features of motor cells both in open and closed pulvini, to identify ultrastructural changes associated with leaflet movement. Pulvini have a central vascular core bordered by thick-walled collenchyma cells, which in turn are surrounded by several layers of cortical parenchyma cells. Cortical motor cells exhibit ultrastructural features similar to those reported in homologous cells of other pulvini. The vacuolar compartment contains two kinds of vacuoles: nontannin vacuoles, which change both in number and size during leaflet movement, and tannin vacuoles, which may act as an ion reservoir. No differences in wall thickness were found between flexor and extensor motor cells. Thick walls of collenchyma cells show numerous pits with plasmodesmata through which the phloem parenchyma cells and the inner cortical motor cells are connected. Tannin vacuoles and calcium oxalate crystals are common inclusions of phloem parenchyma cells. The tissue arrangement and the occurrence of pits with plasmodesmata in the central cylinder cells provide evidence of symplastic continuity through the central cylinder between the extensor and flexor regions of the motor organs. The greater amplitude of Robinia leaflet movements may be related to the extension of motor regions, the scarcity of lignification in the central vascular core, and the thin flexor walls.