Udo Kristen

Membrane Flow via the Golgi Apparatus of Higher Plant Cells

Publisher Summary This chapter discusses the membrane flow via the Golgi apparatus (GA) of higher plant cells. GA plays a significant role in membrane flow processes, as a large part of the intracellular transport of macromolecules takes place via dictyosomes. Therefore, the membranes of the GA are considered as packing material and transportation vehicles for the transfer of polysaccharides and proteins from their sites of synthesis to the plasma membrane (PM). The transfer processes constitute a membrane flow beginning at the GA or, when starting at the endoplasmic reticulum (ER), via the GA. Membrane transfer from the ER to the PM via the GA can be divided into three different phases. Those three phases are, (1) beginning at the ER and ending at the forming faces of the dictyosomes; (2) comprising the flow through the cisternal stack of the dictyosomes, and (3) extention from the maturing faces of the dictyosomes to the PM. The velocity of granulocrine secretion involves measurements based on the use of content markers; measurements based on membrane markers; and calculations derived from a morphometric analysis of thin sections. The recycling of membrane from the PM is a necessary consequence of granulocrine secretion.

Microtubules are involved in maintaining the cellular polarity in pollen tubes ofNicotiana sylvestris

SummaryThe polarity of a growing pollen tube is clearly reflected by a distinct zonation of the cytoplasmic content. The vegetative nucleus and the generative cell (GC) are located in the tip region of the tube, and the basal cytoplasmic portion is highly vacuolated. Using pollen tubes ofNicotiana sylvestris Spegazz. & Comes grown in vitro, we examined the effects of varying concentrations of the microtubule inhibitors colchicine and propham. The depolymerization of the cortical microtubules by 25 μM colchicine led to a disorganization of the cytoplasm, i.e., vacuolization of the tip region, and to a deranged position of both the vegetative nucleus and the generative cell. The same concentration of colchicine inhibited tube growth by 10–20% of the control. Mitosis of the GC was not affected. Only from concentrations of 200 μM the configuration of the GCs microtubules was altered and an inhibition of mitosis was observed. At this concentration the disorganization of the cytoplasm was always reversible, but neither inhibition of mitosis nor derangement of the nuclear positioning was. At 1,800 μM colchicine, pollen tube growth was inhibited by 50% of the control. Using propham, the same three steps of action were observed, although propham proved to be about a hundred times more effective than colchicine. We conclude that the cortical microtubules of the pollen tube are involved in maintaining cellular polarity, probably as a part of a heterogeneous cytoskeletal network including also microfilaments and membranous elements. Nuclear positioning seems to be dependent on both, the tubes cortical and the GCs microtubules. A possible involvement of the extracellular matrix in maintaining intracytoplasmic polarity is suggested.

The anti-microtubule drug carbetamide stopsNicotiana sylvestris pollen tube growth in the style

SummaryRecently, we found that the anti-microtubule drugs colchicine and propham caused the absence of microtubules and thus loss of cytoplasmic zonation in in vitro growing pollen tubes ofNicotiana sylvestris, but did not seriously affect growth. In the present study we used the herbicide carbetamide as an anti-microtubule drug. It had the same effect as colchicine and propham: the cytoplasm, including the generative cell, was no longer concentrated in the tip but was distributed randomly. In addition, ultrastructural investigations have shown that even the vesicle zone, usually found at the very tip of pollen tubes, had disappeared in some tubes. Nonetheless, in vitro growth was not inhibited by more than 20% over a period of 22 h.In contrast, tube growth in plants ceased 1 cm down in the style when carbetamide was applied to the stigma before pollination. At the lowest concentration causing this effect, microtubules of the vegetative cell had disappeared and the cytoplasm was distributed randomly, as it was for in vitro grown tubes. It can be concluded that microtubules of the vegetative cell are essential for pollen tube growth in the style.

Ultrastructure and a possible function of the intercisternal elements in dictyosomes.

The slime-producing dictyosomes in the placentary papillae of Aptenia cordifolia (L.f.) Schwant. show some structural peculiarities: (1) the number of their cisternae is conspicuously large in comparison with those of other cormophyta; (2) the spaces between the extremely flat vesicle-producing cisternae of the maturing face are considerably higher than those between the other cisternae; (3) the intercisternal elements show a pearl-string form rather than a fibrillar form-especially on tangential sections. Based on personal and on cited findings, the following hypothesis is developed: The intercisternal elements effect the compression of the central region of the secretory cisternae. This causes the production of vesicles to remain restricted to the marginal region of the cisternae, even if these cisternae contain hypertonic or soaking substances.

Membrane flow in plants: Fractionation of growing pollen tubes of tobacco by preparative free-flow electrophoresis and kinetics of labeling of endoplasmic reticulum and golgi apparatus with [3H]leucine

SummaryTobacco (Nicotiana tabacum L.) pollen, germinated 4 hours in suspension culture, was labeled with radioactive leucine and fractionated into constituent membranes by the technique of preparative free-flow electrophoresis. Tubes were ruptured by sonication directly into the electrophoresis buffer. Unfortunately, the Golgi apparatus of the rapidly elongating pollen tubes did not survive the sonication step. However, it was possible to obtain useful fractions of endoplasmic reticulum and mitochondria. To obtain Golgi apparatus, glutaraldehyde was added to the homogenization buffer during sonication. Plasma membrane, which accounted for only about 3% of the total membrane of the homogenates as determined by staining with phosphotungstate at low pH, was obtained in insufficient quantity and fraction purity to permit analysis. Results show rapid incorporation of [3H]leucine into endoplasmic reticulum followed by rapid chase out. The half-time for loss of radioactivity from the pollen tube endoplasmic reticulum was about 10 minutes. Concomitant with the loss of radioactivity from endoplasmic reticulum, the Golgi apparatus fraction was labeled reaching a maximum 20 minutes post chase. The findings suggest flow of membranes from endoplasmic reticulum to the Golgi apparatus during pollen tube growth.

Die Morphologie der Schleimsekretion im Fruchtknoten vonAptenia cordifolia

SummaryDuring seed development the gynaeceum ofAptenia cordifolia produces a mucilage rich in carbohydrates and protein. The mucilage-producing placentary papillae are analyzed in different developmental stages by electron microscopy. Just before mucilage production is started, the rough ER occurs but sparsely. At that time the dictyosomes are inconspicuous and of low activity. When mucilage production commences, one can observe hypersecretory dictyosomes and ER-ensheathed vacuoles (storage vesicles) as the main structural components. It is suggested that the complexes of rough ER and probably fused Golgi vesicles are the synthetizing units of the carbohydrate protein mucilage, since in these complexes both components can be identified cytochemically. Fusion sites of plasmalemma and vesicles indicate processes of exocytosis-probably involving the Golgi apparatus. In addition, a holocrine excretion of the mucilage initially enclosed in the storage vesicles via degeneration of the protoplast is assumed.ZusammenfassungDer Fruchtknoten vonAptenia cordifolia enthÄlt wÄhrend der Samenentwicklung einen proteinreichen Polysaccharidschleim. Verschieden alte schleimproduzierende Placentarpapillen werden einer elektronenmikroskopischen Analyse unterzogen. Kurz vor dem Einsetzen der Schleimproduktion ist das rauhe ER noch spÄrlich entwickelt. Der Golgi-Apparat ist unauffÄllig und wenig aktiv. Zu Beginn der Schleimbildung sind als hauptsÄchliche Strukturkomponenten hypersekretorische Dictyosomen und ER-umschlossene Vakuolen (storage vesicles) zu beobachten. Es wird angenommen, da\ diese Komplexe aus rauhem ER und vermutlich mitèinander verschmolzenen Golgi-Vesikeln die charakteristischen Synthese-Einheiten für den Polysaccharid-Protein-Schleim darstellen, da sie nachweislich neben Polysacchariden auch Proteine enthalten. Membranfusionen zwischen Vesikeln und dem Plasmalemma deuten auf Exocytose-Prozesse unter Beteiligung des Golgi-Apparates hin. Daneben wird eine holocrine Ausscheidung des in den storage vesicles zunÄchst gespeicherten Polysaccharid-Protein-Schleimes bei Degeneration des Protoplasten vermutet.

Pentachlorophenol affects mitochondria and induces formation of golgi apparatus-endoplasmic reticulum hybrids in tobacco pollen tubes

SummaryThe toxic effect of pentachlorophenol (PCP) on the growth and ultrastructure of tobacco pollen tubes was tested using a semivivo technique of tube culture. In this technique the pollen tubes were allowed to grow in the pistilin situ for 24 hr before they protruded from the cut end of the style and came into contact with the medium containing PCP. The inhibitory effect of different PCP concentrations was determined by measuring the length of tube bundles. The intracellular action of PCP was analysed by electron microscopy. This biocide caused four obvious alterations in the pollen tube ultrastructure: (1) swelling of the mitochondrial saccules; (2) enlargement of the dictyosomes by the increase of the cisternal diameter and the number of cisternae per stack; (3) formation of cup-shaped Golgi apparatus-endoplasmic reticulum hybrid structures (GER hybrids) showing continuities of ER and Golgi cisternae; (4) formation of stacked and/or concentric arrangements of rough ER cisternae. It is suggested that swelling of saccules was directly due to the uncoupling of oxidative phosphorylation whereas the changes of the endomembrane system were caused by energy depletion due to the inhibition of ATP synthesis. These changes are consistant with dynamic concepts of dictyosome and ER function when membrane formation exceeds membrane use in the production of secretory and transition vesicles. Thus, the enlargement of the dictyosomes and the formation of GER hybrids are thought to result from inhibition of budding of vesicles from the Golgi apparatus or from both the ER and the Golgi apparatus, respectively.

Granulocrine Ausscheidung von Narbensekret durch Vesikel des Endoplasmatischen Retikulums beiAptenia cordifolia

SummaryDuring anthesis the young stigma papillae ofAptenia cordifolia produce a slime containing polysaccharides and proteins. Among other details of the ultrastructure there are chloroplasts during their transformation into chromoplasts, and, as the most conspicuous item, numerous vesicles with fibrillar contents, measuring 0,5–1,5 μm in diameter, partly fused with each other. Some vesicles are directly connected with the intracisternal space of the ER, other vesicles are merged with the plasmalemma. The Golgi apparatus is developed but weakly, and shows no hypersecretory activity. Because of these observations a transport and an exocytotic extrusion of the secretion substances via ER vesicles is concluded.ZusammenfassungDie jungen Narbenpapillen vonAptenia cordifolia scheiden während der Blühzeit ein polysaccharid- und proteinhaltiges Sekret aus. Die Feinstruktur zeigt u. a. Chloroplasten vermutlich während der Metamorphose zu Chromoplasten und als auffälligste Komponente zahlreiche 0,5–1,5 μm große, z. T. miteinander verschmolzene Sekretvesikel mit fibrillärem Inhalt. Einige dieser Vesikel stehen mit der intrazisternalen Phase des ER in direkter Verbindung, andere fusionieren mit dem Plasmalemma. Der Golgi-Apparat ist nur schwach entwickelt und weist keine hypersekretorische Aktivität auf. Auf Grund dieser Befunde wird angenommen, daß der Sekrettransport und die exocytotische Ausschleusung des Sekrets durch ER-Vesikel erfolgen.

Development and disintegration of phragmoplasts in living cultured cells of a GFP∷TUA6 transgenic Arabidopsis thaliana plant

Summary. Cultured suspension cells of Arabidopsis thaliana that stably express a green-fluorescent protein–α-tubulin 6 fusion protein were used to follow the development and disintegration of phragmoplasts. The development and disintegration of phragmoplasts in the living cultured cells could be successively observed by detecting the green-fluorescent protein fluorescence of the microtubules. In the early telophase spindle, where two kinetochore groups and two daughter chromosome groups had completely separated from one another, fluorescence appeared in the interzone between the two chromosome groups. The fluorescent region was gradually condensed at the previous equator and increased in fluorescence intensity, and finally it formed the initial phragmoplast. The initial phragmoplast moved from the cell center towards the cell periphery, and it lost fluorescence at its center and became double rings in shape. The expansion orientation of the phragmoplast was not always the same as that of the future new cell wall before it came in contact with the cell wall. The phragmoplast did not usually come in contact with the cell wall simultaneously with its entire length. A portion of the phragmoplast which was earlier in contact with the cell wall disappeared earlier than other portions of the phragmoplast. The duration of contact between any portions of the phragmoplast and the plasma membrane of the cell wall was 15–30 min. The fluorescence intensity of the cytoplasm did not seem to be elevated by the disintegration of the strongly fluorescent phragmoplast.

Ultrastructural responses of tobacco pollen tubes to heat shock

SummaryThe effect of elevated temperatures on semivivo growth and ultrastructure of tobacco pollen tubes was investigated. Tube growth was decreased by about 50% at 35 °C, independent of the duration of treatment, and at 40 °C and above there was no growth of tubes. Heat treatment caused ultrastructural changes like accumulation of membranous materials, concentric stacking of rough endoplasmic reticulum, reduction in vesicle production by dictyosomes, increase in the fenestrated regions of the Golgi cisternae, swelling of mitochondrial saccules and increase in the electron density of the mitochondrial matrix. Furthermore, the dictyosomes of the treated tubes showed significant increase in the number of cisternae from 30 to 45 °C. The temperature induced changes were persistant at least for 24 h in 35 °C grown pollen tubes. The possible reasons for the tube growth inhibition are discussed on the basis of the ultrastructural alterations caused by elevated temperatures.