Satellite-Rich DNA in Cucumber: Hormonal Enhancement of Synthesis and Subcellular Identification

Abstract

Cucumber hypocotyl DNA in neutral CsCl distributes into a mainband comprising 59% of the total, and two large satellite bands which contribute 41%to the DNA pattern. Organelle enrichment studies show that the densities of mitochondrial and chloroplast DNA coincide with those of the satellite bands. At least 12-19% of total cucumber DNA is associated with the cytoplasmic organelles. These In prior studies, gibberellin-induced elongation of the cucumber hypocotyl was correlated with an increased incorporation of [3H]thymidine into DNA (1). Autoradiographic and fractionation experiments (2) further suggested that much of this synthesis might be extranuclear, reflecting a hormoneenhanced multiplication of chloroplasts in the elongating zone (3). This possibility has now been investigated at the level of different DNA density peaks by CsCl gradient centrifugation. In this regard, several higher plants, including cucumber, have previously been shown to contain particularly large amounts (up to 44%) of satellite DNAs in neutral CsCl (4-8). These large plant satellites-in analogy with the satellite DNAs of animal cells (9)-have variously been reported to be rapidly renaturing (4,7,8,10), associated with the genes for rRNA (4, 11) and of nuclear origin (7,10,12). The relationship between cytoplasmic organelle DNA and the large plant satellites, however, was not seriously investigated.In the present report we demonstrate that a considerable fraction of the large satellite DNAs in cucumber are associated with chloroplasts and mitochondria. We further show that in elongating hypocotyl tissue the rates of synthesis of organelle-rich satellite DNA and mainband DNA respond in vivo to hormone treatments by increasing markedly. Alternatively (method 2), hypocotyl sections were processed as described by Tautvydas (17) except that steeping was carried out at 0-2°in 4% gum arabic, 0.4 M sucrose, 6 mM Mg acetate, 0.3 mM dithiothreitol, and 5 mM morpholinoethane sulfonate, pH 6.4. Resuspension buffer was without gum arabic and contained 0.15% 2-ethyl-1-hexanol. Method 3 was identical to method 2 except that 0.1% Triton X-100 was included during the steeping procedure. For the isolation of chloroplasts the filtered homogenate was centrifuged at 1000 X g, and the pellet was dispersed in modified Honda medium supplemented with 40 mM Mg+2 and incubated with DNase (70 ug/ml) at 0°for 15-20 min. After addition of 0.1 M EDTA, discontinuous sucrose gradient centrifugation was carried out (2). For the isolation of mitochondria filtered homogenate was centrifuged at 5000 X g for 5 min and the supernatant was recentrifuged at 23,500 X g for 20 min.The 23,500 X g pellet was treated with DNase as described for chloroplasts and then rinsed in Tris-saline-EDTA.

Chromosome, DNA and Plant Evolution

“…One of the hazards associated with isolation of satellite DNA by the CsCI density gradient method is the possibility of contamination with DNA derived from chloroplasts and mitochondria (Kadouri et al, 1975). Possibly, much of the DNA of Cucurbitaceae identified by some workers as satellite may be of this origin.…”

Gibberellic Acid Enhancement of DNA Turnover in Barley Aleurone Cells

“…ii Uptake cpm~~~~~DNA cpm/mg DNA (19). The total DNA content of isolated aleurone layers does not increase during incubation, although thymidine is incorporated into DNA.…”

Distinction between Nuclear Satellite DNAs and Chloroplast DNA in Higher Plants

“…The suggestion of Kadouri et aL (7) that the percentage of chloroplast DNA should be highest in plants with nuclei of low DNA content is probably correct, but their value of 12 to 19%o chloroplast DNA in cucumber does not agree with these results. The cucumber genome is not unusually small as suggested by Kadouri et aL (7). Also, not all plant species with small genomes have satellite DNA (e.g.…”

“…Recently it was suggested that a major component of certain satellite DNAs was organellar (3,7). This implied that the method commonly used for nuclear DNA preparation, the selective solubilization of organelles by treatment with Triton X-100 (10), was ineffective, and that chloroplast DNA constituted a much greater proportion of the total DNA than was previously supposed (11).…”