DNA SYNTHESIS, CELL DIVISION, AND CELL DIFFERENTIATION DURING LEAF DEVELOPMENT OF XANTHIUM PENNSYLVANICUM

Leaf growth consists of two basic processes, cell division and cell enlargement. DNA synthesis is an integral part of cell division and can be studied with autoradiographic techniques and incorporation of some labeled precursor. Studies were made on the synthesis of nqclear DNA through incorporation of 3H-thymidine in various parts of the lamina during the entire course of leaf development of Xanthium pennsylvanicum. The time course analysis of DNA synthesis was correlated with cell division and rates of cell enlargement. Significant differences in 3H-thymidine incorporation were found in various parts of the lamina. Cell division and DNA synthesis were highest in the early stages of development. Since no 3H-thymidine was incorporated after cessation of cell division (LPI 2.8) in the leaf lamina, it appears that DNA synthesis is not needed for enlargement and differentiation of Xanthium cells. Rates of cell enlargement were negligible in the early development and reached their maximum after cessation of mitoses, between plastochron ages (LPI) 3 and 4. Cells matured between LPI's 5 and 6. Enzymatic activity was correlated with cell division and cell differentiation at various stages of leaf development. LEAF DEVELOPMENT has been studied (Avery, 1933; Foster, 1936; Esau, 1965; Denne, 1966) primarily from the morphological point of view, but the metabolic aspects related directly to development are experimentally more difficult to approach and are less well understood. The application of radioisotopes and autoradiographic techniques (Jensen, 1962; Rogers, 1967) has opened new avenues of approach in biochemical studies of development. The purpose of this investigation is to present a quantitative description of cell division and 3H-thymidine incorporation into nuclear DNA during the whole course of leaf development. Since 3H-thymidine is a precursor utilized in nucleic acid synthesis, it is hoped that pertinent conclusions can be drawn concerning DNA synthesis as affected by various developmental stages. An attempt also is made to correlate DNA synthesis and cell division with cellular differentiation, emphasizing their quantitative and temporal aspects. MATERIALS AND METHODS-Burs of Xanthium pennsylvanicum Wallr. (Synonym X. saccharatum) were germinated in flats of soil. After the first two foliage leaves had developed, the seedlings were transplanted in pots. The plants were grown in a walk-in type chamber where the light intensity varied around 700 ft-c. Temperature was adjusted to 25 C i 0.5. The relative humidity 1 Received for publication 8 December 1969. This investigation was supported by National Science Foundation research grant GB-6635. The authors are indebted to the Rev. R. C. Shurer, O.S.A. for his help in photomicrography. was maintained at 50 i 3 %. To maintain vegetative growth, the illumination was cycled to give a light period of 18 hr and a dark period of 6 hr. Leaves on the primary shoot were numbered in order of their appearance with studies being made on the middle portion of leaves eight and nine. The morphological ages of the leaves were determined by use of the leaf plastochron index (LPI). Leaves selected for this experiment ranged between LPI 4.0 and 6.0. The duration of one LPI was 3.4 days under these conditions. For estimation of the LPI reference is made to Erickson and Michelini (1957) and Erickson (1960). Tritiated thymidine (10 lc/ml sp. act. 6.7 c/mM) was introduced by foliar absorption of the isotope. The whole shoot of a Xanthium plant with the desired leaf was placed in 0.1 % Tween 20 for 15 min to facilitate the uptake of the isotope (Juniper, 1959). The wetting agent was then replaced by the radioisotope for a period of 2-4 hr. Air was bubbled into the medium to increase oxygen concentration. After two or four hours of growth in 3H-thymidine the treated leaf was removed from the radioisotopic solution and rinsed with distilled water. The tissue was sectioned at 4,u, stained with Feulgen, and coated with NTB-2 Kodak Nuclear Tract Emulsion (Jensen, 1962; Rogers, 1967). The slides were exposed to the emulsion for 2 weeks at 4 C. A Bausch and Lomb Whipple micrometer was used for grain counts and estimation of the percentage of labeled nuclei in the vein, near-vein, and lamina regions, and throughout the entire lamina. A series of experiments were performed to ensure the specificity of 3H-thymidine incorporation into