Arya K. Bal

Selective staining of the ultra-structural components of cortical granules and golgi cisternae of sea urchin eggs.

SummaryPhosphotungstic acid (PTA) was found to stain one of the components (dark bodies) of the cortical granules in sea urchin eggs. In the oocytes, where miniature presumptive cortical granules have been shown to be formed near Golgi complexes, the materials of the Golgi cisternae also revealed PTA-positive properties. Loss of PTA stainability after hyaluronidase treatment suggests mucopolysaccharide nature of this material. These observations moreover, strengthen the hypothesis that Golgi complexes are involved in the formation of cortical granules.

Ultrastructural changes in the accessory-cells and the oocyte surface of the sea urchin Strongylocentrotus dröbachiensis during vitellogenesis

SummaryElectron microscopic observations on ripening ovaries of Strongylocentrotus dröbachiensis, have revealed evidence for micropinocytotic activity of the oocyte surface during early vitellogenesis stages. The microvilli that cover the surface of the oocyte, have been shown to engulf glycogen particles, derived from the accessory-cells. At these early vitellogenesis stages massive globulated accessory-cells are in close contact with the oocyte. Later during the development of the oocyte, the accessory cells retract from the oocyte surface and become considerably shrunken into thin cytoplasmic sheets. Evidence presented here also throws some light on the dynamics of glycogen transfer, from the accessory cell to the oocyte. Furthermore, the ultrastructure of the accessory-cell has been clarified with special reference to the formation and disintegration of globules. Although glycogen in the accessory-cells is organised in the form of alpha particles, during and after transfer into the oocyte, it is only visualised in the beta and gamma forms. This investigation demonstrates clearly the nutritive role of the accessory-cells during oocyte differentiation in sea urchins.

Seasonal changes in carbohydrates of perennial root nodules of beach pea

Seasonal changes in amyloplast, starch, total soluble sugars, non-reducing sugars and reducing sugars of perennial root nodules of beach pea (Lathyrus maritimus) were studied. Ultrastructural changes in nodule cells of beach pea indicated an accumulation of large amounts of amyloplasts with multiple starch grains in summer months. As the winter season sets in, degradation of amyloplasts and starch grains was detected. The membranes of amyloplasts faded out in winter and the structure of the amyloplasts was lost. The degradation of starch grains showed some electron-dense fiber-like material and amorphous structures. Quantitative data revealed an increase in starch reserves during the summer and depletion during the winter. Total soluble sugar and non-reducing sugar concentrations peaked in the middle of the winter, whereas reducing sugar concentrations showed an increase in the fall. These results indicate that elevated levels of various sugars most likely help to maintain high osmolarity of cells so that the dormant nodules do not freeze during the prolonged periods of cold in the winter.

Endoplasmic reticulum activity and cell wall breakdown in quiescent root meristems of Allium cepa L.

Summary Dissolution of cell wall material has been recorded in the cortical and provascular elements of the quiescent root meristems of Allium cepa L. bulbs. The cell walls disappear, resulting in the formation of masses of cytoplasm with free floating nuclei. Ultrastructural observations indicate elaboration of massive rough endoplasmic reticular elements, which directly participate in the breakdown process.

Localization of Plant Lipids for Light Microscopy Using P-Phenylenediamine in Tissues of Arachis Hypogaea L.

p-Phenylenediamine (pPD) can be used en bloc to preserve and differentiate cell lipids in aldehyde-fixed peanut plant tissues treated with osmium tetroxide during dehydration in 70% ethanol. Semithin plastic sections for light microscopy need no further staining and can be mounted in Histoclad after drying on a slide. Brown staining above background differentiates lipid-containing structures. Nonspecific staining can be distinguished in control preparations extracted en bloc with lipid solvents.

Asymbiotic association of Rhizobium with pea epicotyls treated with a plant hormone.

Treatment of epicotyls of dark-grown pea (Pisum sativum L.) seedlings with indole-3-acetic acid causes swelling of the tissue. Application of Rhizobium to the cut surface of the swollen tissue results in the development of an “infection”. The infection spreads in the cortical cells and proceeds 2–3 mm deep into the stem within 3–4 days. An acetylene reduction assay used for detecting nitrogen-fixation capacity of the infected tissue was negative at 10% [O2]; however, if [O2] was reduced to below 1%, some activity could be detected. Ultrastructural observations indicate that the cytoplasmic contents of the infected cells are destroyed and no membrane structure around the bacteria is formed during this infection. Rhizobium does not appear to have developed any symbiotic relationship with the host. Failure to develop symbiosis appears to result in a parasitic or saprophytic association and the nitrogen fixed under such conditions may not be of any use to the plant.

Cell wall (outer membrane) of bacteroids in nitrogen-fixing peanut nodules

Rhizobia in peanut nodules are transformed from rod-shaped cells to extremely large spherical bacteroids. The invading rhizobia shed their outer membranes soon after their release into the host cells. The outer membranes are seen to be peeled off and replaced by new outer membranes before rhizobia are differentiated into nitrogen-fixing bacteroids. Evidence is presented that peanut bacteroids, in spite of their spheroplast-or protoplast-like appearance, do possess both the inner and the outer membrane.

Histochemical detection of in vitro DNA polymerase activity.

SummaryA significant level of nuclear DNA polymerase activity (deoxyribonucleoside-triphosphate: DNA deoxyribonucleotidyltransferase EC 2.7.7.7) can be detected in (a) cryostat sections and (b) toluene treated tissues of onion seed radicles by autoradiography with 3H-TTP. Incorporation was optimal in the presence of all four deoxyribonucleoside triphosphates and dithiothreitol but sensitive to (a) DNase (b) high concentrations of KCl and (c) the sulfhydryl reagents p-chloromercuribenzoate and N-ethyl maleimide. Attempts to activate incorporation with the inclusion of (a) small concentrations of DNase and (b) ATP in the incubating medium failed. The percentage of cells displaying enzyme activity in vitro was similar to the number observed in vivo suggesting that incorporation was associated with S-phase cells and is genuine DNA replication as opposed to repair synthesis.To our knowledge, an histochemical demonstration of in vitro DNA polymerase activity has not been reported before.

Seasonal changes in oleosomic lipids and fatty acids of perennial root nodules of beach pea.

Seasonal changes in the fatty acid composition of phospholipids (PL), monoglycerides (MG), diglycerides (DG), free fatty acids (FA) and triglycerides (TG) separated from oleosomes (lipid bodies) of perennial root nodules of beach pea (Lathyrus maritimus) were analysed. Thin layer chromatography (TLC) revealed that PL and MG are the major lipids in nodule oleosomes. The fatty acid profile and overall double bond index (DBI) varied among lipid classes depending upon the season. High DBI in PL and MG found during late winter and early spring indicated that they may play a major role in winter survival and regeneration of perennial nodules. The DBI of DG was high at the end of the fall season and the DBI of FA and TG was high in summer months. The dominant fatty acids are C16:0 followed by C18:0 and C18:1. The levels of many unsaturated fatty acids such as C18:1, C18:2 and C18:3 increased while saturated fatty acid C18:0 decreased during winter. These unsaturated fatty acids possibly play an important role in the protection of nodule cells from cold stress. Nodules seem to retain some fatty acids and selectively utilize specific fatty acids to survive the winter and regenerate in spring.

Pre- and Postwinter Changes in the Root Nodules of Lathyrus maritimus (L.) Bigel. with Special Reference to Storage Organelles

Histological and ultrastructural studies of the root nodules of naturally growing beach pea (Lathyrus maritimus [L.] Bigel.), an arctic/subarctic legume from the shorelines of Newfoundland, revealed considerable seasonal variation in the presence of storage organelles, such as amyloplasts and oleosomes (lipid bodies). Three successive years of samples taken during late autumn and early spring were processed for both light and electron microscopy. After fruiting, large numbers of amyloplasts with starch grains and oleosomes were observed in the uninfected interstitial cells and parenchyma cells of the nodule tissues. These storage organelles could not be seen in the cells of nodules sampled during postwinter periods before aerial shoots emerged, indicating their importance in overwintering. Persistent infection threads with rhizobia could be seen, and rod-shaped rhizobia in senescent cells were indicative of reversion of bacteroids to rod forms within the nodule tissue.