Björn Walles

Immunogold Localization of Pectin and Callose in Pollen Grains and Pollen Tubes of Brugmansia suaveolens — Implications for the Self-Incompatibility Reaction

Summary Pollen of Brugmansia suaveolens was grown in vitro or in vivo after compatible or incompatible pollination. Ultrathinsections of fixed and embedded specimens were treated with immunogold label for ultrastructural localization of acidic pectins (monoclonal antibody JIM 5), methyl-esterified pectins (monoclonal antibody JIM 7) and callose (anti-(1-3)-β-glucan antibody) in order to elucidate the distribution and intracellular pathway of the cell wall constituents_ Pectins, present in the intine of the pollen grain and in the outer layer of the bilayered pollen tube wall, seem to be synthesized in the dictyosomes activated upon germination. Their intracellular transport from the dictyosomes to the pollen tube tip, the site of fusion with the cell wall, occurs in fibrillar or bipartite particles that contain pectins in a highly methyl-esterified form, but no callose. In both intine and pollen tube cell wall a radial gradient of pectin esterification is observed. Pectins are less esterified with increasing distance from the plasma membrane. Considering the fact that pectins are secreted in a highly esterified form, the gradient indicates the presence of enzymatic activity, causing pectin de-esterification during cell wall development. Upon incompatible pollination both layers of the pollen tube wall undergo thickening. Pectinaceous particles are deposited on the inside of the cell wall or seemingly get stuck during migration through the inner callosic layer. Whereas pectinaceous secretory vesicles in compatible pollen tubes show a high grade of esterification, during the incompatibility reaction pectic aggregates of low esterification grade appear in the cytoplasm. This might indicate the presence of de-esterifying enzymes in the secretory vesicles.

Electron microscope studies on cell differentiation in synchronized cultures of the green algaScenedesmus

SummaryThe normal ultrastructural changes during the cell cycle have been studied in synchronized cultures ofScenedesmus obtusiusculus Chod. grown in a complete nutrient solution. The interphase cell has a three-layered wall, an oblong chloroplast with a central pyrenoid, a dictyosome associated with the nuclear envelope and surrounded by a cylinder of endoplasmic reticulum (amplexus), a microbody adjacent to the pyrenoid, and a centriole pair.The cell passes two or three mitotic divisions followed by a simultaneous wall formation around the daughter cells (autospores). The nuclear divisions are preceded by reproduction of the dictyosome and the centriole pair and presumably also of the chloroplast. Microtubules originate during prophase and persist until the autospores are formed.It was found that the organelles occupy definite positions during the different stages of the cell development.

DNA FRAGMENTATION AND NUCLEAR DEGRADATION DURING PROGRAMMED CELL DEATH IN THE SUSPENSOR AND ENDOSPERM OF VICIA FABA

In Vicia faba, both the suspensor and the endosperm are short‐lived structures. The aim of this work was to elucidate and compare programmed cell death (PCD) mechanisms in these two ephemeral tissues. To achieve this, we used the TdT‐mediated dUDP fluorescent (FITC) nick end labeling (TUNEL) technique to trace DNA fragmentation and transmission electron microscopy to follow chromosomal and nuclear degradation. The TUNEL experiments demonstrated DNA fragmentation in the endosperm nuclei 13 d after pollination (dap) and in the suspensor at 14 dap. However, the ultrastructural studies did not show any chromosomal degradation in the nuclei of the suspensor or endosperm until 17 dap, indicating that the DNA fragmentation is an initial step in the PCD pathway. We have further documented fundamental differences in the degeneration process of the nuclei of the two tissues. In the suspensor the heterochromatin becomes more condensed during degeneration and disperses to the nuclear periphery as electron‐dense areas. The nucleolus will keep its round and condensed shape for some time before expanding into an irregular body. In the endosperm the heterochromatin is more like a network throughout the nucleus, and the nucleolus will eventually split into pieces scattered inside the heterochromatin. Internal vesicle‐like structures appear in the nuclei of the suspensor at 11–13 dap. However, they might have a communicative function not necessarily related to the cell death. We conclude that both the suspensor and the endosperm go through PCD processes, but the pathways leading to dismantling of the cells do not follow identical routes in the two tissues. DNA fragmentation occurs in intact cells and within an intact nuclear envelope and not in cells that already are damaged. PCD in the endosperm precedes PCD in the suspensor, indicating that they might receive different signals or that the signal triggers different internal death programs in the two tissues.

Localization and release of allergens from tapetum and pollen grains ofBetula pendula

SummaryAlthough intact pollen grains are assumed to be the primary carrier of pollen allergens, specific immunoreactive components have been found in other aerosol fractions, e.g., starch grains and remains of tapetal cells Cryo-scanning-electron-microscopy results demonstrate the presence of a clear network of strands connecting the tapetum with the microspores. The distribution of protein in tapetal orbicules, pollen wall, and pollen cytoplasm was tested by histochemical stains for light microscopy and transmission electron microscopy. The protein is mainly localized at the apertures and starch grains in the cytoplasm of pollen and in the core and on the surface of tapetal orbicules. Monoclonal antibodies Bv-10, BIP3, and BIP4 have been used to locate the cellular sites of pollen and tapetal allergens inBetula pendula (syn.B. verrucosa). The application of rapid-freeze fixation prevented relocation of allergens from their native sites. The allergens are predominantly found in the starch grains and to lesser extent in the exine. We also tested interactions between mature birch pollen and human fluids: saliva, nostrils fluid, and eyes solution. The aim was to mimic more closely the in vivo situation during allergenic response. In all cases we observed several pollen grains that were burst and had released their cytoplasmic contents. In the nose the allergens are released from the pollen within minutes. In rhinitis, nasal pH is increased from the normal pH 6.0 to 8.0. When we used nasal fluid at pH 8.0, the number of ruptured pollen grains increased. The mechanism that might induce formation of small allergen-bearing particles from living plant cells is discussed.

Functional Anatomy of the Ovule in Broad Bean, Vicia faba L. II. Ultrastructural Development up to Early Embryogenesis

Ovules of broad beans (Vicia faba) have been studied to analyze ultrastructural features for nutrient transport to the embryo sac at various ontogenetic stages up to 10 d after pollination. In unpollinated flowers a notable homogeneous or fibrillar material is deposited in the endostome, between the two integuments and on the nucellus. Osmiophilic globules accumulate at the plasmalemma and in the walls at the micropylar end of the inner integument. These globules increase in number after fertilization and appear also in other cells near the embryo sac. The central cell, which has some wall ingrowths typical for transfer cells, shows intrusive growth between cells of the nucellar cap. After fertilization wall thickenings occur in cells close to the embryo sac. At 10 d after pollination the inner integument has degenerated entirely. Also the nucellus, including the nucellar cap, is digested. In the endosperm free-nuclear divisions start and the cytoplasm increases in amount. Wall ingrowths are formed along the whole embryo sac boundary. The suspensor consists of two pairs of multinucleate cells: the pair adjacent to the embryo proper have rounded cells; the other pair have elongated ones. The suspensor cells that are attached to the embryo sac boundary become transfer cells. Their plastids have prolamellar bodies, and these structures are not seen anywhere else in the ovule. Our study confirms that transfer cells are common at junctions between the different generations in the ovules, that the transport to the embryo sac is apoplastic, and that symplastic transport is possible between endosperm and embryo and further between suspensor and embryo proper.

On the presence of plastids and the eyespot apparatus in a porfiromycin-bleached strain ofEuglena gracilis

SummaryUltrastructural studies were performed on a strain ofEuglena gracilis bleached with the antibiotic porfiromycin. A limited number of organelles could be identified as plastids by their possession of a double envelope and a stroma containing bunches of minute thylakoids, DNA-fibrils and ribosomes. A stigma was situated close to the reservoir. Opposite the stigma a paraflagellar body was observed in the main flagellum.

Chromoplast development in a carotenoid mutant of maize

SummaryThe lethal recessive mutantlycopenic in maize is characterized by the synthesis of lycopene instead of the normal carotenoids. At normal conditions of illumination it loses chlorophyll by photo-oxidation. Seedlings of this mutant and of normal maize were grown at light intensities of 25–30 lux and 500–30,000 lux. Their plastid development was studied by electron microscopy.At low light intensities a kind of mesophyll chloroplast with elongated grana, long unpaired thylakoid segments, and sometimes prolamellar bodies is formed in mutant plants. In corresponding bleached plants the plastids are transformed into chromoplasts containing characteristic lycopene crystalloids similar to those found in tomato fruits. Various stages in this chromoplast development are described and illustrated. Also bundle-sheath plastids were found to develop into chromoplasts.It is concluded that the ultrastructure of plastids in a tissue is influenced by the nature of their pigments and that an altered carotenoid composition therefore can give rise to development of chromoplasts in plants which normally lack such organelles.

Microsporogenesis inPinus sylvestris L. VIII. Tapetal and late pollen grain development

This last portion of our developmental study ofPinus sylvestris L. pollen grains extends from just prior to the first microspore mitosis to the microsporangial dehiscence preparatory to pollen shedding. In nine years of collecting each day the duration of the above period was 7 to 11 days. Tapetal cells extended into the loculus and embraced microspores during the initial part of the above period. Thereafter tapetal cells receded, became parallel to parietal cells and so imbricated that there appeared to be two or three layers of tapetal cells. Tapetal cells were present up to the day before pollen shedding, but only rER and some mitochondria appeared to be in good condition at that time. A callosic layer (outer intine) was initiated under the endexine before microspore mitosis. After the first mitosis the first prothallial cell migrated to the proximal wall and was covered on the side next to the pollen cytoplasm by a thin “wall” joining the thick outer intine. There are plasmodesmata between pollen cytoplasm and the prothallial cell. After the second mitosis the second prothallial cell became enveloped by the outer intine. The inner intine appears after formation of the two prothallial cells but before the third mitosis. During this two-prothallial cell period before the third mitosis, plastids had large and complex fibrillar assemblies shown to be modified starch grains. After the third mitosis plastids of the pollen cytoplasm contained starch and the generative cell (antheridial initial), the product of that mitosis, is enveloped by the inner intine. On the day of pollen shedding cells are removed from the microsporangial wall by what appears to be focal autolysis. The tapetal and endothecial cells for 10–15 µm on each side of the dehiscence slit are completely removed. One or more epidermal cells are lysed, but both a thin cuticle and the very thin sporopollenin-containing peritapetal membrane remain attached to the undamaged epidermal cells bordering the dehiscence slit. Our study terminates on the day of pollen shedding with mature pollen still within the open microsporangium. At that time there is no longer a clear morphological distinction between the outer and inner intine but, judging by stain reactions, there is a chemical difference. The exine of shed pollen grains was found to be covered by small spinules on the inner surface of alveoli. These had the same spacing as the Sporopollenin Acceptor Particles (SAPs) associated with exine initiation and growth.

Functional anatomy of the ovule in broad bean(Vicia faba L.). I: Histogenesis prior to and after pollination

Structural adaptations promoting apoplastic transport to the embryo sac were studied in broad beans (Vicia faba) during histogenesis from ovule primordia to seed. Nucellus and the inner integument disappear at an early stage. The outer integument then adjoins the embryo sac boundary. In the thin part distal to the funiculus the cells of the persisting integument are smaller than in other parts and develop PAS-positive wall ingrowths opposite the embryo. At the late globular stage, the embryo establishes contact with the embryo sac boundary. In the contact zone, embryo cells develop wall ingrowths. Wall ingrowths are further formed on both sides of that part of the endosperm that is inserted between the outside of the cotyledons and the embryo sac boundary. The wall proliferations characterize all these cells as transfer cells. We conclude that the embryo sac is supported with nutrients from digested maternal tissues (nucellus, chalaza, inner integument, and part of the outer integument). These tissues are generally rich in starch grains. After the embryo has established contact with the embryo sac boundary, it is supported by transport of solutes from transfer cells in the outer integument.

Structural studies of pollen tube growth in the pistil of Strelitzia reginae

SummaryFor this work we have used various microscopical methods (LM, SEM, and TEM) to study pollen tube growth and interaction with the transmitting tisse inStrelitzia reginae, which has an open style. By the use of SEM it was possible to trace the exact route of the pollen tubes in the ovary of this plant and demonstrate that they exclusively follow the outlines of the transmitting tissue. The average rate of pollen tube growth through the style was 1.8 mm h−1. The most significant effect of the pollination was a thickening of the distal wall of the subepithelial cells in the style. A secretion covers the stigma and the ovarian transmitting tissue and fills the stylar canal. This exudate contains lipids, polysaccharides, and proteins.