Histochemical studies of alpha and beta-glucosidases in the germinating pollen grains ofPortulaca grandiflora

The paper deals with the distribution of alpha and beta-glucosidases in the germinating pollen grains ofPortulaca grandiflora. Both these enzymes are localized in the pollen wall and the cytoplasmic granules. The latter are distributed throughout the pollen cytoplasm, pollen tube and stigma hair. In non-germinating pollen grains, enzymes are concentrated in the pollen wall. Stigma hair sheaths are completely free from enzymes. The functional significance of these enzymes in the hydrolysis of phenolic glycosides and polysaccharides is discussed.     

Histochemical studies on the distribution of 5-nucleotidase in the germinating pollen grains ofEschscholtzia californica cham.

The present paper incorporates a detailed study on the distribution of 5-nucleotidase in the germinating pollen grains ofEschscholtzia califomica. The intense activity of this enzyme has been found in the wall of pollen grains and pollen tubes and small positive granules in the lumen of both in the pollen grains and pollen tubes. The presence of this enzyme in the wall is presumably connected with the permeability and transport process and growth regulation of the pollen tubes.     

Histochemical demonstration of non-specific esterase in wheat root

Azo-coupling methods were used for demonstrating non-specific esterase in the wheat root in all parts of a transverse section, usually with the exception of the woody parts of the vascular bundle. The central cylinder gave a more intense reaction than the primary cortex and the rhizoderm. The reaction was not inhibited by dodecyl sulphate. A weakening of the reaction intensity was observed after application of AgNO₃. The Tween method did not yield reliable results.     

DNA uptake during electroporation of germinating pollen grains

Electroporation of germinating pollen grains of Nicotiana gossei (L.) Domin under a variety of conditions showed that DNA was taken up by the pollen without detrimental effects on the viability of the pollen. By optimizing both the field strength of the electroporation pulse and the DNA concentration in the electroporation medium up to 6% of the donor DNA can be taken up by the germinating pollen while maintaining a pollen viability of 90%. Field strengths as high as 9 kV/cm could be applied to germinating pollen grains without detrimental effects on viability. Southern hybridizations demonstrated that DNA encoding the marker enzyme β-glucuronidase (GUS) was incorporated into electroporated pollen. Germinating pollen, treated in this manner, is capable of producing 300–400 seeds per capsule of viable seed when applied to the stigmas of compatible flowers of N. gossei which has been emasculated 4 days earlier.     

Transgenic antibiotic resistance may be differentially silenced in germinating pollen grains

Transgenic tobacco (Nicotiana tabacum L.) plants, carrying the neomycin phosphotransferase (NPT II) gene from E. coli, are resistant to kanamycin when grown from seeds on kanamycin containing medium. Tissue and cell cultures derived from those transformants also express resistance and regenerate complete plantlets in the presence of the antibiotic. This unspecific response to the selective condition has led to the belief that the foreign gene is continuously active or uniformly inducible in all cells of the transgenic plant. However, our experiments show that this view is not true for pollen grains during in vitro germination. Pollen grains isolated from kanamycin resistant tobacco plants carry and transmit the foreign gene but do not express resistance when germinating in vitro. This data presents evidence for differential silencing of a foreign gene in a mature gamete. On the other hand, immature pollen grains (microspores) appear to express resistance. The point of the downregulation of the neomycin transferase gene during pollen maturation is discussed.Abbreviations kan kanamycin sulfate - NPT II neomycin phosphotransferase II - sr streptomycin sulfate     

Histochemical studies on protease formation in the cotyledons of germinating bean seeds

Summary Protease formation in Phaseolus vulgaris L. cotyledons during seed germination was studied histochemically using a gelatin-film-substrate method. Protease activity can be detected by this method on the 5th day of germination, at approximately the same time that a rapid increase of activity was observed by a test-tube assay with casein as a substrate. At the early stage of germination, protease activity was observed throughout the cotyledon except in two or three cell layers below the cotyledon surface and in several cell layers around the vascular bundles. A highly active cell layer surrounding the protease-inactive cells near the vascular bundles is suggested to be a source of the protease.Brooklyn Botanic Garden Contribution No. 202.     

Histochemical and biochemical studies on esterase activity in the rat ovary

A correlative histochemical and biochemical study has been made of the changes in esterase positive sites in immature (10-, 20- and 30-days old), mature normal cycling (3-, 5- and 8-months-old), pregnant and lactating rat ovaries. The typical perivascular esterase-positive sites localized in the hilar portion, branch along the blood vessels and traverse into medullary and cortical portions of the ovary. The stromal vascularity surrounding the normal developing follicles, corpora lutea, atretic follicles and interstitial gland tissue showed rich activity of this enzyme system. On semiquantitative basis the number, intensity and quantity of esterase-positive sites vary with the maturation and reproductive states of the rat. The administration of estradiol-17 beta increased the fine perifollicular and theca externa perivascular esterase-positive sites, whereas atropine and reserpine affected severely both the large and fine meshwork of esterase-positive sites. Biochemical estimates of acetylcholine esterase activity endorse these histochemical observations. The possible roles of AChE activity in varied ovarian functions are discussed.     

The behavior of nuclei in germinating pollen grains of wheat,rice and maize

Theoretical and Applied Genetics - 1. Three species of Gramineae were used for obeservations on the behavior of the tube nucleus and sperm nuclei in germinating pollen grains ofTriticum aestivum,...     

Carboxypeptidases of germinating triticale grains

Presence of five carboxypeptidases was found in endosperm of germinating triticale grains, while two of them in scutellum. Changes of their activities during four days of germination suggest that carboxypeptidase II plays an important role at initial stage of germination, while carboxypeptidases I and III - at subsequent stages of the process. High activity of carboxypeptidase II both in scutellum and endosperm of dry grains accompanied by its decrease during germination, and on the other hand, the appearance of carboxypeptidases I and III activities at the 2^nd and 3^rd day of the process seems to confirm such functions of these enzymes. Experiments with GA₃ indicated that carboxypeptidase I was synthesized in scutellum, and carboxypeptidase III — in aleurone layer. Carboxypeptidases I and II cleave N-CBZ-Phe-Ala, and carboxypeptidase III — N-CBZ-Ala-Met and N-CBZ-Ala-Phe as substrates with the highest rate.     

tRNA synthesis in germinating pollen

The synthesis of tRNA was demonstrated in pollen ofNicotiana tabacum L. according to the incorporation of labeled uracil, adenosine and guanosine during 4 h of germination. tRNA was extracted from postribosomal supernatant and purified by polyacrylamide gel electrophoresis. The incorporation of guanosine was about 1.68 times higher than that of adenosine. This finding indicates that the whole tRNA chain is formed in pollen tubes.     

The effect of cherry leaf roll virus infection on the performance of birch pollen and studies on virus replication in germinating pollen

The rates of germ tube elongation in vitro of pollen from cherry leaf roll virus (CLRV)-infected birch (Betula pendula) did not differ significantly from those of pollen from virus-free trees. Differences in percentage germination of pollen collected from trees at different sites were significant but percentages of germination of pollen from virus-infected and virus-free trees did not differ greatly from one another. In vivo, pollen from infected trees germinated on healthy and CLRV-infected stigmas and callose plugs formed in both types of tissues. However, the extent of callose plug formation was greater in the pollen tubes of virus-free grains germinating in infected stigmas than in reciprocal crosses. CLRV coat antigen was detected by ELISA in stigmatic tissue, from healthy trees, on which virus-carrying pollen grains had germinated. Using fluorescein isothiocyanate conjugated to CLRV-specific γ-globulin, viral coat antigen was detected throughout germ tubes from virus-carrying but not virus-free pollen germinating in vitro. Protoplasts released following Driselase digestion of pollen germ tubes from virus-infected trees contained CLRV antigen detectable by ELISA. During germination of virus-infected pollen there was little synthesis of viral coat protein or nucleic acid but, following inoculation with purified virus particles, protoplasts made from healthy germinating pollen produced increasing amounts of CLRV-specific antigen implying that CLRV replicated in this system.     

Histochemical demonstration of neurotoxic esterase

We developed a histochemical method for localizing neurotoxic esterase (NTE), defined as the phenylvalerate (PV)-hydrolyzing esterase that is resistant to 40 microM paraoxon (A) but inactivated by paraoxon plus 50 microM mipafox (B). NTE is considered to be the target enzyme in the production of organophosphorus ester-induced delayed neurotoxicity (OPIDN). Cryostat sections were incubated in a medium containing alpha-naphthyl valerate and 6-benzamido-4-methoxy-m-toluidine diazonium chloride (fast violet B) after treatment with the above-mentioned inhibitors, leading to formation of an aqueous insoluble precipitate at sites of enzymatic activity. NTE activity was estimated as staining detectable in A but not in B. In the central nervous system (CNS) of chicken, NTE appeared to be present primarily in the somata of most neurons, but at sites indistinguishable from those of the other inhibitor-resistant and -sensitive alpha-naphthyl valerate-hydrolyzing esterases. It could not be distinguished in the CNS of cat, probably because it constitutes less than 3% of the total PV-hydrolyzing activity in the CNS of that species.     

The role of alpha-glucosidase in germinating barley grains

The importance of α-glucosidase in the endosperm starch metabolism of barley (Hordeum vulgare) seedlings is poorly understood. The enzyme converts maltose to glucose (Glc), but in vitro studies indicate that it can also attack starch granules. To discover its role in vivo, we took complementary chemical-genetic and reverse-genetic approaches. We identified iminosugar inhibitors of a recombinant form of an α-glucosidase previously discovered in barley endosperm (ALPHA-GLUCOSIDASE97 [HvAGL97]), and applied four of them to germinating grains. All four decreased the Glc-to-maltose ratio in the endosperm 10 d after imbibition, implying inhibition of maltase activity. Three of the four inhibitors also reduced starch degradation and seedling growth, but the fourth did not affect these parameters. Inhibition of starch degradation was apparently not due to inhibition of amylases. Inhibition of seedling growth was primarily a direct effect of the inhibitors on roots and coleoptiles rather than an indirect effect of the inhibition of endosperm metabolism. It may reflect inhibition of glycoprotein-processing glucosidases in these organs. In transgenic seedlings carrying an RNA interference silencing cassette for HvAgl97, α-glucosidase activity was reduced by up to 50%. There was a large decrease in the Glc-to-maltose ratio in these lines but no effect on starch degradation or seedling growth. Our results suggest that the α-glucosidase HvAGL97 is the major endosperm enzyme catalyzing the conversion of maltose to Glc but is not required for starch degradation. However, the effects of three glucosidase inhibitors on starch degradation in the endosperm indicate the existence of unidentified glucosidase(s) required for this process.     

A comparative study on the distribution of non-specific esterase amongst the various constituents of retinas of some vertebrates

A wide coverage of the retinae of a large number of animals (Calotes, Varanus, Naja, Athene, Passer, Streptopelia, Psittacula and Funambulus) from the point of view of the histoenzymological distribution of non-specific esterase amongst the various constituents reveals mostly identical patterns. They are as follows: 1. Outer segments - positive in all cases. 2. Outer plexiform layer - equipped with enzymatic activity in all the instances. 3. Inner nuclear layer - thin cytoplasmic rim of the neurons characterized by positive activity; the nuclei of the neurons are completely negative. 4. Inner plexiform layer - this layer is endowed with the enzymatic activity. 5. Ganglion cells - negative in all cases. 6. Nerve fibres of the layer of nerve fibres, situated adjasent to ganglion cells are positive in all the animals; in case of squirrel oligodandroglial cells present in the region have demonstrated activity of a high order. On of the high-lights of the present contribution is demarcation of the inner plexiform layer into three stratified zones, equipped with enzymatic activity in Calotes, Streptopelia, Naja and Funambulus. Such stratifications are not seen in Varanus, Passer and Psittacula. The significance of the various patterns and the equipment of the enzyme in various constituents at various locals have been discussed in relation to the metabolic functions, zone-wise and interzone-wise in visual processes of various animals.     

Histochemical studies of pollen: storage pockets in the endoplasmic reticulum (ER)

Summary The pollen grain of cotton (Gossypium hirsutum) was examined histochemically at the light and electron microscope level. The cytoplasm of the pollen contains an unusual storage unit which consists of a pocket of endoplasmic reticulum (ER) containing lipid droplets and dictyosome vesicles. The ER pockets are large enough to be seen with the light microscope if thin enough sections are used (0.3–1.5). The results of the histochemical analyses show that the dictysome vesicles are rich in carbohydrate and contain protein and lipid as well. The ER contains large amounts of protein which may be arginine rich. Some carbohydrates may also be present in the ER. The ER is covered with ribosomes so that the pockets are unusually rich storage units containing abundant protein, carbohydrate, lipid and RNA. The light microscope localization of carbohydrates was confirmed by the periodic acid-silver method. Other storage units in the cytoplasm were also studied. A new method for the embedding of plant tissue for thin sectioning for light microscopy is presented.This work was supported by a Public Health Service fellowship 5-F2-GM-22, 031-02 from the National Institute of General Medical Sciences, by NSF grant GB 3460, by NIH grant 5-RO1-CA 0356-10 and by the Miller Institute for Basic Science.