The effect of benzyladenine and thidiazuron on endosperm development and DNA endoreduplication
--L. Schweizer, G. L. Yerk, R.L. Phillips, R. L. Jones and F. Srienc

Cytokinins have been associated with mitotic cell division. Cytokinin levels increase greatly after fertilization and during the periods when there is rapid seed and fruit growth (Bohner and Bangerth, Physiol. Plant. 72:316-320, 1988; Carnes and Wright, Plant Sci. 57:195-203, 1988; Schrieber et al., Plant Physiol., in press, 1993). In maize kernels, the decrease in mitotic activity coincides with the decrease in cytokinin levels (Kowles and Phillips, Int. Rev. Cytol. 112:97-136, 1988; Schrieber et al., Plant Physiol., in press, 1993). Following the reduction in mitotic activity, there is an increase in DNA content per nucleus. This increased DNA content per nucleus has been identified in all inbreds and hybrids examined (Kowles and Phillips, PNAS USA 82:7010-7014, 1985). Endoreduplication of the nuclear DNA is responsible for the observed increases in DNA content per nucleus. These data indicate that there may be a tightly coupled developmental regulation between cell division, DNA endoreduplication, and cytokinin levels in developing kernels.

The purpose of this study was to determine the relationship between cytokinin levels and mitosis and DNA endoreduplication during kernel development. Benzyladenine, an adenine derivative, and thidiazuron (N-phenyl-N'-1,2,3-thiodiazol-5-ylurea), a urea derivative with cytokinin activity were exogenously applied to maize inbred line W64a. This line was chosen for its small seed size, thus alterations in cell number would be more easily detected.

W64a was planted in 1990 and 1991 at St. Paul. All ears were bulk pollinated with W64a pollen on the same day. Each year, four replicates in a randomized complete block, containing eight treatments of exogenously applied cytokinins, were evaluated. The eight treatments were control (not sprayed), control spray (sprayed with dimethylsulfonate (DMSO), a solvent used to solubilize the benzyladenine and thidiazuron), three concentrations of benyzladenine: 10-3M, 10-4M, and 10-5M and three concentrations of thidiazuron: 10-3M, 10-4M, 10-5M. Spray treatments were applied on the leaf subtending the ear until run-off occurred each day from 6 to 10 days after pollination (DAP).

Kernel samples were collected from the middle third of the ear each day from five to twenty-two DAP. Samples were fixed in 3:1 (95% ethanol: glacial acetic acid) overnight. The following day, the samples were transferred to 70% ethanol and stored at -20 C. Squashes of endosperm tissue from the control were made with propionic carmine. At least 1000 cells per endosperm were scored for mitotic activity for each day from 6 to 22 DAP. Mitotic index was calculated as the percentage of mitotically active nuclei among the total nuclei.

Nuclei preparations were made according to the method of Kowles et al. (The Maize Handbook, M. Freeling and V. Walbot, eds., 1993). These preparations were stained with Mithramycin A and analyzed by flow cytometry to determine the DNA content per nucleus and the total cell number per endosperm. In 1990, four replicates per treatment were measured, and in 1991 three replicates per treatment were analyzed. In both years treatments were evaluated each day from 8 to 18 DAP. The following parameters were measured on a per endosperm basis: average DNA content per nucleus, total nuclei number, number of 3C, 6C, 12C, 24C, 48C, and 96C nuclei.

Analysis of variance was performed on the data using a split-split plot design with the treatments being the whole plot factor and heat units being the sub-plot factor. Heat units were utilized rather than days after pollination to standardize the stage of kernel development across environments. The data for the two years were analyzed separately due to heterogeneity of error variances.

Mitotic index peaks at 8 DAP and then declines rapidly. The change in total nuclei number per endosperm over cumulative heat units followed the same pattern in 1990 and 1991, however, the same data taken as a function of days after pollination plateaus at different time points, indicating that the use of heat units to remove environmental effects is appropriate. No significant variation was observed among the treatments for average DNA content per nucleus and 3C nuclei number per endosperm. Significant variation was seen for total nuclei number, and the number of 6C, 12C, 24C, 48C, and 96C nuclei per endosperm. The lowest concentration of thidiazuron increased total cell number as did the benzyladenine treatments. Conversely, the highest concentration of thidiazuron reduced total cell number. These results indicate that cytokinins have an effect on both mitosis, as measured by total cell number per endosperm, and DNA endoreduplication, as measured by the number of 12C, 24C, 48C, and 96C nuclei. The mechanism by which the observed changes in DNA content and cell number occur is unknown. Benzyladenine has been implicated in transcriptional control of gene expression. Although a number of mechanisms have been proposed for the action of thidiazuron on mitotic tissues, a clear model is still unavailable.

Based on these findings, measurement of endogenous cytokinin levels in kernels treated with benzyladenine and thidiazuron at different developmental times is warranted. In particular, measurement of endogenous levels of purine cytokinins and ribonucleoside and ribonucleotide levels which are altered during metabolism of cytokinins, might aid in elucidating the mechanisms by which these compounds act. 


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