Martin Bopp

A cytokinin test with high specificity

SummaryA cytokinin bioassay based on bud formation in 10-cell-long caulonema filaments of Funaria hygrometrica is described. The test has high specificity and sensitivity; is completed in 2 days; exhibits linearity between cytokinin concentration in the medium and bud number; and no buds are formed in the absence of a cytokinin.

Die Entwicklung von Zelle und Kern im Protonema von Funaria hygrometrica Sibth

Zusammenfassung1.Die Entwicklung von Zelle und Kern in den Caulonemen vonFunaria hygrometrica Sibth. wurde untersucht.2.Ein Zusammenhang zwischen bestimmten Funktionen der Zelle und dem Kernzustand in verschiedenen Abschnitten konnte festgestellt werden. So besitzen die Spitzenzellen, auf die das Plasmawachstum beschränkt ist, einen großen, stark “vacuolisierten” Nucleolus. Die Zellen, in denen sich die Membran bräunt, zeigen eine meßbare Kernvergrößerung.3.Chromocentren werden erst in älteren Zellen des Zellfadens beobachtet, sie liegen immer dem Nucleolus außen an. In alten Zellen verschwindet der Nucleolus, infolgedessen bleiben die Chromocentren als größere Chromatinbrocken allein erhalten.4.Zwischen der äußeren Form des Kernes und der Plastiden in den einzelnen Zellen besteht eine weitgehende Übereinstimmung, diese ändert sich sehr regelmäßig von rundem über ovalen zu spindelförmigem Umriß.5.Die Bildung von Tmemen (Trennzellen) und von Seitenfäden werden als inaequale Zellteilungen beschrieben.6.Intercalare Zellteilungen, die durch Isolierung hervorgerufen werden, führen zu gleichwertigen Tochterzellen. Es ist der einzige Fall, bei dem in zurückliegenden Zellen des Protonemas vonFunaria hygrometrica aequale Zellteilungen vorkommen.7.Die Knospenbildung ist nicht durch besondere karyologische Vorgänge gekennzeichnet. Die mit großem Nucleolus versehene Spitzenzelle des Protonemas setzt sich kontinuierlich in der Scheitelzelle der Moospflänzchen fort.

Über die Synthese von DNS in den Keimpflanzen von Sinapis alba L.

SummarySeedlings of Sinapis alba were grown under standard conditions. In the hypocotyls and cotyledons DNA synthesis still takes place 36 h after sowing. This synthesis decreases in the following 24 h, but an incorporation of 3H-thymidine was found 108 h after sowing.Autoradiographic studies demonstrate the incorporation of 3H-thymidine into cell nuclei. While some nuclei are homogeneously labelled, in other nuclei the radioactivity appears preferentially or exclusively in the chromocenters.A transfer into the dark of plants previously grown in light (for 24 h or 48 h) does not result in an increase of DNA-synthesis again.Seedlings of Sinapis alba were grown under standard conditions. In the hypocotyls and cotyledons DNA synthesis still takes place 36 h after sowing. This synthesis decreases in the following 24 h, but an incorporation of (3)H-thymidine was found 108 h after sowing.Autoradiographic studies demonstrate the incorporation of (3)H-thymidine into cell nuclei. While some nuclei are homogeneously labelled, in other nuclei the radioactivity appears preferentially or exclusively in the chromocenters.A transfer into the dark of plants previously grown in light (for 24 h or 48 h) does not result in an increase of DNA-synthesis again.

Markierungsmuster nach H3-Thymidineinbau in Kernen der Hypokotylzellen von Sinapis alba L.

DNA-synthesis in the hypocotyls of Sinapis alba L. was studied with H3-thymidine labelling. Cells from hypocotyl segments were stained by the Feulgen-method and squash preparations were made. The following labelling patterns were observed: 1. Labelling of the chromocentres only. 2. Nuclear area evenly labelled. 3. No radioactivity in the chromocentres. This pattern was rarely seen. — The frequency of the first two types in different tissue segments is not equal. In segments with more differentiated cells there was an increase in the percentage of nuclei with radioactivity only in the chromocentres. This could be due to a prolongation of the phase of synthesis in the chromocentres in this tissue. — The total number of labelled nuclei decreases basipetally as well as with the age of the hypocotyl. In hypocotyls of seedlings older than 52 hrs radioactivity appeared only sporadically in the nuclei. The decrease in the number of labelled nuclei is faster than the decline of the corresponding measurable total DNA synthesis in the hypocotyl. This can either be due to extra nuclear DNA synthesis or depend on an increase in DNA synthesis in the later replicating heterochromatic region of the nucleus.

Streckungshemmung durch FUDR bei den Hypokotylen von Sinapis alba

Seedlings of Sinapis alba which were grown in light for 24, 48 and 72 h after 36 h of dark treatment renew elongation when transferred to dark again. The rate of elongation decreases with increased light treatment. The per cent as well as absolute inhibition of elongation by FUDR decreases with the duration of light treatment. The shortening of the hypocotyl is due mainly to the inhibition of cell elongation. The inhibition is not directly proportional to DNA synthesis at any particular time.Plants without cotyledons are less inhibited than those with cotyledons. Cytosine arabinoside is inhibitory only at high concentrations. According to these results elongation inhibition by FUDR does not involve the entire DNA-synthesis.SummarySeedlings of Sinapis alba which were grown in light for 24, 48 and 72 h after 36 h of dark treatment renew elongation when transferred to dark again. The rate of elongation decreases with increased light treatment. The per cent as well as absolute inhibition of elongation by FUDR decreases with the duration of light treatment. The shortening of the hypocotyl is due mainly to the inhibition of cell elongation. The inhibition is not directly proportional to DNA synthesis at any particular time.Plants without cotyledons are less inhibited than those with cotyledons. Cytosine arabinoside is inhibitory only at high concentrations. According to these results elongation inhibition by FUDR does not involve the entire DNA-synthesis.

Charakterisierung des Regenerationsprozesses im Caulonema eines Mooses durch autoradiographische Bestimmung der DNS-Synthese

Ten-cell-long filaments of the caulonema of Funaria hygrometrica were isolated and labeled with (3)H-thymidine. During the process of regeneration this precursor is incorporated into the nucleus and the chloroplasts. The nuclei of aged cells are preferentially labeled, even the nuclei of such cells which probably will no longer divide.From these facts it is concluded that DNA synthesis can occur during the process of regeneration irrespectively of a following cell division.SummaryTen-cell-long filaments of the caulonema of Funaria hygrometrica were isolated and labeled with 3H-thymidine. During the process of regeneration this precursor is incorporated into the nucleus and the chloroplasts. The nuclei of aged cells are preferentially labeled, even the nuclei of such cells which probably will no longer divide.From these facts it is concluded that DNA synthesis can occur during the process of regeneration irrespectively of a following cell division.

[Experiments in the analysis of the protonema development in mosses : V. The action of kinetin in caulonema regeneration].

1. On isolated caulonema branches of Funaria hygrometrica buds can be induced by kinetin in the same way as on intact protonemata. This is possible even in the case of isolated caulonema composed of only 5 cells. The buds are also formed at the same places as in intact protonemata. 2. The number of buds induced is dependent on the concentration of kinetin and the age at which the caulonemata were isolated. The largest increase occurs when the concentration of kinetin in the culture medium is between 0.1 and 0.5 mg/l. This concentration range is the same for protonemata 13 to 17 days old. More than 1 mg/l kinetin results in no further propagation of buds. The highest number of buds was formed on caulonemata of 19-day-old protonemata. 3. If the isolated caulonemata are at first laid out on agar free of kientin and only after 1 to 10 hrs transferred to kinetin medium, the number of buds decreases in an exponential curve. The half-life is 5 hrs. It is independnet of the age of caulonemata, i.e. of the number of buds formed immediately after the isolation. 4. Inhibition of DNA-synthesis by 5-FUDR has no influence on the regeneration process responsible for the decrease of bud formation or on the induction of buds by kinetin. During regeneration on medium containing FUDR, the number of buds decreases in the same way as in the control. Even when DNA-synthesis and cell division are suppressed for several days, the induction of buds can be demonstrated with Acridine Orange. 5. The experiments support the view that a reaction partner of kinetin is present in caulonema cells which during regeneration to the chloronema stage is either changed in one step so that it cannot react any longer with kinetin or is no longer present.Summary1.On isolated caulonema branches of Funaria hygrometrica buds can be induced by kinetin in the same way as on intact protonemata. This is possible even in the case of isolated caulonema composed of only 5 cells. The buds are also formed at the same places as in intact protonemata.2.The number of buds induced is dependent on the concentration of kinetin and the age at which the caulonemata were isolated. The largest increase occurs when the concentration of kinetin in the culture medium is between 0.1 and 0.5 mg/l. This concentration range is the same for protonemata 13 to 17 days old. More than 1 mg/l kinetin results in no further propagation of buds. The highest number of buds was formed on caulonemata of 19-day-old protonemata.3.If the isolated caulonemata are at first laid out on agar free of kientin and only after 1 to 10 hrs transferred to kinetin medium, the number of buds decreases in an exponential curve. The half-life is 5 hrs. It is independnet of the age of caulonemata, i.e. of the number of buds formed immediately after the isolation.4.Inhibition of DNA-synthesis by 5-FUDR has no influence on the regeneration process responsible for the decrease of bud formation or on the induction of buds by kinetin. During regeneration on medium containing FUDR, the number of buds decreases in the same way as in the control. Even when DNA-synthesis and cell division are suppressed for several days, the induction of buds can be demonstrated with Acridine Orange.5.The experiments support the view that a reaction partner of kinetin is present in caulonema cells which during regeneration to the chloronema stage is either changed in one step so that it cannot react any longer with kinetin or is no longer present.Zusammenfassung1.An isolierten Caulonemaästen von Funaria hygrometrica lassen sich durch Kinetin Knospen induzieren. Dies ist auch noch bei Isolaten möglich, die nur fünf Zellen umfassen. Die Knospen liegen an denselben Stellen, an denen sie auch an intakten Protonemen vorkommen.2.Die Anzahl der induzierten Knospen ist von der Kinetinkonzentration und dem Alter, zu dem die Caulonemen isoliert wurden, abhängig. Die größte Zunahme der Knospenzahl liegt zwischen 0,1 und 0,5 mg/l Kinetin im Kultursubstrat. Dieser Konzentrationsbereich ist für Protonemen vom 13.–17. Tage gleich. Mehr als 1 mg/l Kinetin führt zu keiner weiteren Vermehrung der Knospen. Die meisten Knospen wurden an Caulonemen von 19 Tage alten Protonemen gebildet.3.Werden die isolierten Caulonemen zunächst auf kinetinfreien Agar ausgelegt und erst nach 1–10 Std auf Kinetinsubstrat übertragen, so nimmt die Knospenzahl in einer exponentiellen Kurve ab. Die Halbwertzeit beträgt 5 Std. Sie ist unabhängig vom Alter der Caulonemen, d.h. von der anfänglich gebildeten Knospenzahl.4.Hemmung der DNS-Synthese durch 5-FUDR hat weder einen Einfluß auf den für die Abnahme der Knospenbildung verantwortlichen Regenerationsvorgang noch auf die Knospeninduktion durch Kinetin. Die Knospenzahl nimmt bei Regeneration auf FUDR-haltigem Substrat genau so ab wie bei der Kontrolle. Auch wenn die DNS-Synthese und damit die Zellteilung für mehrere Tage unterdrückt wird, läßt sich die Induktion von Knospen mit Hilfe von Acridinorange nachweisen.5.Die Versuche sprechen dafür, daß in den Caulonemazellen ein Reaktionspartner der Kinetins vorhanden ist, der bei Regeneration zum Chloronemastadium durch einen Schritt so verändert wird, daß er entweder nicht mehr auf Kinetin reagieren kann oder nicht mehr vorhanden ist.

Hemmung der Induktionsvorgänge bei Wurzelhalsgallen durch 2-Thiouracil und 5-Bromuracil

SummaryThe effect of 5-bromouracil and 2-thiouracil onAgrobacterium tumefaciens, onKalanchoe daigremontiana and on the crown galls formed onKalanchoe under the influence ofAgrobacterium has been investigated and the following observations have been made.1.100 to 200 ψ/cm3 of bromo- or thiouracil scarcely inhibit the growth of the bacteria, although the substances, are taken up by the bacteria.2.By a concentration of 20 ψ/cm3 of bromouracilKalanchoe daigremontiana is neither affected in its wound reaction nor in its development. Thiouracil disturbs the development of the lamina after longer periods of action.3.The formation of crown galls is inhibited by both bromo- and thio-uracil, the extent of inhibition being dependent on the concentration. Time experiments showed that bromouracil is effective only within the first 3 days after the infection, whereas thiouracil inhibits the growth of the tumors during their entire development.4.Bromouracil inhibits the tumor cells during the same time as do higher temperatures. It seems probable that pyrimidine and an increase in temperature interfere with the same processes during the transformation of the cells.5.Since it has been shown by numerous investigators that bromouracil is incorporated into DNA, the experiments indicate that the transformation to tumor cells is connected with an increased new formation of a specific DNA.

Zellwandregeneration und Peroxidase-Isoenzym-Synthese isolierter Protoplasten von Nicotiana tabacum L.@@@Cell-wall and peroxidase-isoenzyme synthesis in isolated protoplasts of Nicotiana tabacum L.

SummaryMesophyll-protoplasts of tobacco show increasing peroxidase-activity immediately after isolation. This is due to an enhancement of activity of the constitutive isoenzymes of GIII (=slow migrating cathodic group) and to a new formation of GII-isoenzymes (=slow migrating anodic group). (GII is not present in intact leaves.) As both processes are inhibited by actinomycin and actidion it is assumed that there is a new synthesis of peroxidase enzymes —The peroxidase reaction is independent of the further development of the protoplasts, as was evidenced by culturing protoplasts in different media which regulate the development of the protoplasts. Peroxidase reaction is always the same whether or not there is cell-wall synthesis and cell division. This leads to the conclusion that peroxidases in this case have no relation to synthesis of primary cell-walls. On the other hand they could be related to the dedifferentiation processes that always take place in isolated protoplasts.In the protoplasts GII is localized in the cytoplasma as GIII is, because GII appears before cell walls are synthesised and there is no lack of GII isoenzymes when protoplasts are remazerated after having formed new cell walls.GI (fast migrating anodic group), which is not detectable in isolated protoplasts, appears again after small calluses have developed out of protoplasts. Therefore as far as function is concerned GI is quite different from GII and GIII. The results confirm the hypothesis that GI is localized in intercellular spaces only. It is discussed whether all of the isoenzymes of peroxidase detectable in crude extracts are cytoplasmic ones.Mesophyll-protoplasts of tobacco show increasing peroxidase-activity immediately after isolation. This is due to an enhancement of activity of the constitutive isoenzymes of GIII (=slow migrating cathodic group) and to a new formation of GII-isoenzymes (=slow migrating anodic group). (GII is not present in intact leaves.) As both processes are inhibited by actinomycin and actidion it is assumed that there is a new synthesis of peroxidase enzymes -The peroxidase reaction is independent of the further development of the protoplasts, as was evidenced by culturing protoplasts in different media which regulate the development of the protoplasts. Peroxidase reaction is always the same whether or not there is cell-wall synthesis and cell division. This leads to the conclusion that peroxidases in this case have no relation to synthesis of primary cell-walls. On the other hand they could be related to the dedifferentiation processes that always take place in isolated protoplasts.In the protoplasts GII is localized in the cytoplasma as GIII is, because GII appears before cell walls are synthesised and there is no lack of GII isoenzymes when protoplasts are remazerated after having formed new cell walls.GI (fast migrating anodic group), which is not detectable in isolated protoplasts, appears again after small calluses have developed out of protoplasts. Therefore as far as function is concerned GI is quite different from GII and GIII. The results confirm the hypothesis that GI is localized in intercellular spaces only. It is discussed whether all of the isoenzymes of peroxidase detectable in crude extracts are cytoplasmic ones.

Neue Vorstellungen zum Problem der Isoperoxidasen anhand der Trennung durch Disk-Elektrophorese und isoelektrische Fokussierung@@@New concepts in the study of isoperoxidases based on the separation by disk electrophoresis and isoelectric focusing

SummaryIsoelectric focusing (IEF) of peroxidases of different organs and tissues of Nicotiana tabacum L. was performed on thin-layers of Sephadex and polyacrylamide. Isoelectric points (pIs) of peroxidase bands were measured by special electrodes. — The two types of layers showed very similar results. Reproductibility of pIs was better on polyacrylamide. This method is also easier to practise and requires less time than IEF on Sephadex (3 h versus 18). Thus for analytical purposes the acrylamide-technique is preferable, but if it is necessary to regain the separated enzymes it is better to perform IEF on Sephadex. — When IEF-patterns of peroxidase are compared with the disk electrophoresis (DE) patterns of the same tissue, important differences are observed. The 6 bands of GI (fast migrating, anodic group of DE) are reduced to 2 on the strongly acidic side of IEF (independent of the tissue studied). That means only 2 proteins in GI can be separated by pIs of the molecules. Maybe the heterogeneity of GI bands after DE indicates the presence of conformational isomers (conformers). — Because of the reduced number of bands in GI after IEF there is no difference in the patterns of many tissues (flowers, leaves, shoots, pith) as there is after disk electrophoresis. In the case of GII (slow migrating, anodic group of DE) on the other hand, there are always 4 different bands after isoelectric focusing in the lower acid region instead of 3 after disk electrophoresis. Disk electrophoresis of peroxidase-groups separated by isoelectric focusing shows the same patterns as direct disk electrophoresis of the extract. The methods produce no artifacts. —Comparison of these results with the peroxidase-patterns of tobacco found by other workers and by other techniques leads to the conclusion that there exist at least 4 “isoenzymes” of peroxidase corresponding to the 4 groups GI, GII, GIII, and GIV.