Noriko Nishinari

Relationship between cell division and endogenous auxin in synchronously-cultured tobacco cells

In the cultured tobacco cell, we succeeded in obtaining a partial synchronization of cell division by a combination of pre-starvation, rhythmic light-dark pre-treatment and air tight pre-conditioning. The mitotic index increased during the light period according to the time interval after the end of pre-treatment, and reached its maximum (max=12%) at about 2.5 hr of irradiation, and about 80% of cells completed division 1.5 hr thereafter.During this period, IAA was biosynthesized in the cells, though these cells had been cultured in Murashige and Skoogs medium with 1 mg/l of 2,4-D as a growth substance. IAA was identified by paper chromatography, followed byAvena curvature test and combined gas chromatography-mass spectrometry. The time course of the increase and decrease in the amount of free IAA was parallel to that of the mitotic index. On the other hand, bound IAA increased later and decreased gradually after the end of cell division. Free IAA may have an important role in mitosis.

Cell-free biosynthesis of cytokinins in cultured tobacco cells

Summary Cell-free biosynthesis of free cytokinins not related to the degradation of t-RNA was demonstrated by using enzyme preparation extracted from cultured tobacco ( Nicotiana tabacum cv. Xanthi) cells. The reaction mixture for cytokinin biosynthesis contained 20 mM Tris-HCl buffer (pH 7.6), 5 mM magnesium acetate, 0.5 mM isopentenylpyrophosphate, 50 μ M adenosine as a substrate and dialyzed enzyme extract. After 2.5 hours of reaction at 27°C, N 6 -( Δ 2 -isopentenyl)adenosine, N 6 -( Δ 2 -isopentenyl)adenine and trans-zeatin were biosynthesized. The radioactivity of [U- 14 C]adenosine was incorporated into these three cytokinins after 30 minutes of reaction in the cell-free system. From the time course of biosynthesis of cytokinins and the incorporation of [U- 14 C]adenosine into cytokinins, we concluded that adenosine accepts the isopentenyl group directly to form N 6 -( Δ 2 -isopentenyl) adenosine. This is converted to trans-zeatin via N 6 -( Δ 2 -isopentenyl)adenine by several enzymes.

Increase in activity of IAA-synthesizing enzymes prior to cell division

During the course of cell division in synchronously dividing cultured tobacco cells, the activity of IAA-synthesizing enzymes increased first, then the amount of endogenous IAA, and finally, the number of dividing cells. Based on the results obtained, we discussed the role of endogenous IAA in cell division.