Bernd Knoop

Multiple DNA contents in the haploid protonema of the mossFunaria hygrometrica sibth

SummaryCytofluorometry with fluorochrome “Hoechst 33258” shows that caulonema cells in the protonema of the haploid mossFunaria hygrometrica endoreduplicate their DNA up to 8 or 16 C during normal development. The increase in DNA content depends on age and position of the cells in the filament and occurs in those cells, which normally do not undergo any further growth or division.Single cells from different positions between the tip and the tenth cell of the filament were isolated and regenerated to new protonema. Groth and differentiation of these regenerates depend on the age and position of the original cell. Cytofluorometry of the tip cells of these regenerates shows, that the higher DNA content is maintained during regeneration.

Viability factors in plant suspension cultures: some properties

Summary Spent tissue culture media contain viability factor(s) (VFs) which promote the viability of cells plated at suboptimal density. It was the objective of this study to compare these factors to classical conditioning factors (CFs) reported in previous literature. For this purpose log phase tobacco cells at low density were embedded in agarose drops and covered with conditioned medium, fractions of such medium or fresh medium, respectively. After 3 days of incubation at 25 °C in darkness the percentage of surviving cells was determined. Biological, chemical and physical characteristics of the VF(s) have been analyzed and compared with those of CF(s). It is shown that the VF(s) share many properties with the classical CF(s) supporting our working hypothesis, that VF(s) and CF(s) are similar or identical molecules, namely oligosaccharides or their derivatives.

An Oligosaccharide Growth Factor in Plant Suspension Cultures: A Proposed Structure

Summary Media isolated from growing cell cultures (conditioned media) contain «viability factors» which promote the survival of plant cells plated at low density. We developed a procedure for the purification of the viability factor (VF) of tobacco. Separated by HPLC, a peak of VF-activity correlated with a peak of refractive index. Assuming that the R I -peak is only caused by the VF, its concentration in the conditioned medium is between 10 -8 -10 -9 M. We found that the viability factor is a branched oligosaccharide with a molecular weight of about 1,000 Da. It consists of a «backbone» of three α-1,4-glycosidic-linked galacturonic acids, at least two non-reducing ends (a β-linked glucose and an α-1,2/1,3 or 1,4-linked fucose) and an unidentified monomer, which is positively charged at pH 4. The pectic backbone is responsible for the negative charge at pH 9.5 and probably for its acid lability. The purification procedure which purifies the VF of tobacco almost to homogeneity was also applicable to the VF of carrot. Therefore both factors, from species of different families, have a similar structure. The only difference found was that the VF of carrot is about 200 Da bigger than the VF of tobacco. No structural similarities with other oligosaccharides could be found.

On the action mechanism of cytokinins in mosses: Caulonema specific proteins.

Soluble proteins extracted with Tris-buffer pH 8.8 from both differentiation stages of the protonema of Funaria hygrometrica (chloronema and caulonema) were separated by microgel electrophoresis. 4x10-3 mg protein/gel was applied. The caulonema, which is the only part of the protonema able to form buds following cytokinin treatment, contained 3 protein bands, which were absent in chloronema. We designate them as “caulonema specific proteins” (CSP, approximate molecular weight 500,000). The CSP disappear when the caulonema is isolated and its cells regenerate to chloronema. The CSP bind kinetin (6 Furfurylamino [8-14C]purine) or BAP (6-benzylamino[8-14C]purine) about 10 times stronger than the remaining protein bands in the gel. The greatest part of the cytokinin is metabolized in a short time and consequently a part of the activity in the gel is incorporated into RNA and removable with RNase. Simultaneous application of adenosine and cytokinin reduced the incorporation of radioactivity into RNA and enhanced the specific activity in one of the CSP bands. In all other bands it remained unchanged.From the results it can be suggested that the CSP are probably involved in the early reactions to cytokinin of the target cells.

Regeneration of protonema with multiple dna content from isolated protoplasts of the mossFunaria hygrometrica

SummaryProtoplasts can only be produced from chloronema and the youngest cells of caulonema. After formation of cell walls on a plasmolytic medium protoplasts develop into protonemas on a regular Knop-medium. 6–11 % of the regenerating protonemas however are smaller than the control and show an equally irregular growth, one protonema out of 200 remains exceptionally small. Cytofluorometry with fluorochrome “Hoechst 33258” proves the smaller prctonemas to have a higher DNA content than the control with highest frequencies appearing at the 2 C and 4 C level. These protonemas therefore derive from diploid or tetraploid protonema cells.

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.

Viability Factors in Plant Cell Suspension Cultures - A Novel Bioassay

Summary Factor(s) promoting the viability of cells plated at suboptimal density were found in spent tissue culture media. A rapid bioassay for these factor(s) is described and optimized, and the relationship to the classical conditioning factors is discussed.

Untersuchungen zum Regenerationsmechanismus bei Funaria hygrometrica SIBTH. II. Zur Bedeutung der RNA- und Proteinsynthese bei der Regeneration isolierter Caulonemen

Summary The first sign of regeneration in excised filaments of the moss Funaria hygrometrica consists in intercalarous cell divisions without any cell growth. This process takes place in normal rates even under the influence of inhibitors of RNA- and protein synthesis, while growth of the tip cells and cytokinin induced budding decrease. It is concluded, that the induction of regeneration up to the first division is undependent from a genetic control or long living m-RNA.

[3H]Thymidin-Einbau und DNA-Synthese bei Funaria hygrometrica

SummaryIncubation of caulonema filaments of the moss Funaria hygrometrica with [3H]methylthymidine followed by autoradiography fails to show the real amount of DNA-synthesis. Especially nuclei of steadily growing tip cells remain unlabelled, whereas basic cells become radioactive. This effect is not based on different permeabilities. Autoradiographic results of experiments with 5-fluordesoxyuridine and cycloheximide lead to the conclusion that there exists a marked thymidine-kinase gradient along the filament which depends on age and position of the cells.

Untersuchungen zum Regenerationsmechanismus bei Funaria hygrometricaSibth.: III. Auslösung durch Inhibitoren und Unterdrückung der apikalen Dominanz

Summary The maintenance of cell differentiation in the caulonema of the moss Funaria hygrometrica is a phenomenon of apical dominance. Only filaments with tip cells, growing at a minimum velocity remain unchanged. If this growth rate is not achieved, intercalarous cell divisions occur, representing an early step of regeneration. One way of initiation in intact plants is the application of inhibitors of protein synthesis. They stop growth and make the tip cells send a signal to the basic cells (missing regulator?). Although it can be shown that protein synthesis is inhibited in basic cells too, they respond with intercalarous cell division. Short time treatment with FdUrd gives similar results. The classic method by killing tip cells must include also the side branches, to induce divisions at rates corresponding to those obtained by chemical decapitation. These results are discussed also in view of earlier findings.