Bromodeoxyuridine DNA fiber technology in plants: replication origins and DNA synthesis in tobacco BY-2 cells under prolonged treatment with aphidicolin.

Newly synthesized DNA can be observed on chromosomes or extended chromatin fibers after incorporation and immunodetection of bromodeoxyuridine. This technique, frequently used in animal cells, was adapted for use in BY-2 cells. For the first time, the origins of replication in plant cells could be visualized and monitored on DNA fibers without the use of radioactive traces. The replicon size for BY-2 cells was estimated to be 12.9 microm; and the fork rate, 1.17 microm/h. These values are comparable to those reported for tomato and mustard cells. Furthermore, the data confirm our previous observation that DNA synthesis is not totally blocked by aphidicolin. Bromodeoxyuridine incorporation into DNA was obvious from 24 h onwards after treatment with aphidicolin.

From hormone signal, via the cytoskeleton, to cell growth in single cells of tobacco.

Cultured mesophyll protoplasts of Nicotiana tabacum L. can be hormonally induced into different developmental pathways. In a medium containing auxins (NAA) and cytokinins (BAP) cells divide and eventually give rise to calli. When only auxins are present cells elongate and finally differentiate into very long tubular cells. We focused on the sequence of events leading to elongation. When cultured in a high (1 mg/l) auxin concentration elongating cells seem to pass a certain threshold and increase their nuclear DNA up to about 16C. Cells cultured in a low (0.065 mg/l) auxin concentration only have C-values up to 4C, are unable to pass this threshold and finally fail to elongate. Besides the concentration dependence of the auxin signal, the efflux of auxin seems to be necessary for elongation since addition of TIBA drastically reduces the amount of elongating cells. Concomitant with the changes in nuclear physiology, auxin-induced axiality is seen as sequential rearrangements of microtubules and actin-filaments and of cell wall cellulose microfibrils from 'randomly' arranged in spherical cells to an orientation perpendicular to the long axis of elongating cells.