NUCLEAR-DNA CONTENT IN DIFFERENTIATED TISSUES OF SUNFLOWER (HELIANTHUS-ANNUUS L)

Scanning cytophotometry following Feulgen-staining was used to determine nuclear DNA content in many differentiated tissues of nine cultivars, hybrids or selfed lines of Helianthus annuus. Apart from such ephemeral tissues as endosperm and anther tapetum, it was found that tissue differentiation in sunflower occurs in the diploid condition, cells being arrested in the DNA presynthetic phase (G 1). In certain cases, however, the nuclear DNA content of differentiated G 1 cells does not exactly match the 2C DNA content found in meristematic cells, but may be either higher or lower. In endosperm and anther tapetum cells, nuclear DNA content may be as high as 24 C and 32 C, respectively. Cytological and autoradiographic analyses after 3H-thymidine incorporation reveal that polyploidy in the tapetal cells is due to chromosome endoreduplication. No detectable difference between male-fertile and male-sterile plants exists as far as occurrence and level of cell polyploidy are concerned. The results are discussed in the context of previous investigations on the nuclear condition of differentiated Helianthus annuus tissue

EXTRA DNA-SYNTHESIS IN THE DE-DIFFERENTIATING CELLS OF VICIA-FABA ROOTS

Cell dedifferentiation was induced in Vicia faba root tissues by removing the whole root meristem (decapitation) and the behaviour of the nuclear DNA in the dedifferentiating cells was studied by means of cytophotometric and autoradiographic analyses. Cytophotometric determination after Feulgen-staining showed that: 1. the vast majority of nuclei in differentiated cells were in the DNA postsynthetic phase, but their Feulgen absorption was lower than that of DNA postsynthetic nuclei (G 2, 4 C) in the meristem; 2. such a Feulgen absorption was detected in certain nuclei after root decapitation; 3. all the mitoses in the dedifferentiating tissues were diploid, fully matching the Feulgen absorption of mitoses in the meristem. After 3H-thymidine ( 3H-T) feeding of the decapitated roots and autoradiography, the following results were obtained: 1. two populations of labeled nuclei, characterized by two different levels of scattered labeling occurred in dedifferentiating tissues, slightly labeled nuclei being much more numerous than heavily labeled nuclei; 2. the percentage of labeled nuclei was much greater than that of DNA presynthetic nuclei in the root tissues; 3. almost all the mitoses were labeled after a 16-hour 3H-T feeding; 4. the percentage of slightly labeled nuclei paralleled that of dedifferentiating cells; 5. the duration of the DNA synthesis phase and that of the gap between completion of DNA synthesis and mitosis differed in heavily and slightly labeled nuclei; 6. all nuclei which entered DNA synthesis also entered mitosis. These results are interpreted to mean that: 1. after decapitation, two different DNA syntheses occur in the dedifferentiating root tissues of V. faba: DNA reduplication in cells which dedifferentiate starting from a DNA presynthetic nuclear condition (heavily labeled nuclei) and extra DNA synthesis in cells which dedifferentiate starting from a DNA postsynthetic nuclear condition (slightly labeled nuclei); 2. extra DNA synthesis is required in these dedifferentiating cells for entry into mitosis

LOCATION OF HEITZ ZERSTAUBUNGSSTADIUM (DISPERSION PHASE) IN THE MITOTIC-CYCLE OF PHASEOLUS-COCCINEUS AND THE CONCEPT OF ANGIOSPERM ENDOMITOSIS

DNA microdensitometry and autoradiography after treatment with 3H-thymidine were used to study the phase of dispersion of chromocenters (Z phase) in parallel with chromocentric nuclei in Phaseolus coccineus. In all materials studied, two types of chromocentric nuclei were present. In radicle apices of dry seeds, two classes of nuclear DNA contents were measured, 2 C (G 1) and 4 C (G 2). The 2 C DNA class comprised all chromocentric type I nuclei, the 4 C class included Z phases and chromocentric type II nuclei. The 4 C (G 2) condition of Z phases implies that Z phases maintain their nuclear structure for some time after the end of DNA replication. Shoot apices also contain 2 C (G 1) and 4 C (G 2) nuclei but 4 C nuclei (Z phases and chromocentric type II nuclei) are rare. In seedling root apices, Z phases are from 1.02 to 4.08 times as frequent as prophases. This excludes that Z phase is a very early prophase. DNA microdensitometry shows that the chromocentric type I includes 2 C (G 1) nuclei and nuclei in the first part of the S phase, Z phases include 4 C (G 2) nuclei and nuclei in the last stage of the S phase and chromocentric type II includes mainly 4 C (G 2) nuclei and nuclei in the second part of S. After 90 minutes of treatment with 3H-thymidine all Z phase nuclei are labeled. This result and the microdensitometric data unequivocally demonstrate that Z phase is located at the end of S. The present results and those of previous authors on Z phase are discussed in relation to Geitler's concept of Angiosperm endomitosis. It is concluded that the term "Angiosperm endomitosis" must be abandoned and substituted by the term "chromosome endoreduplication"

CYTOPHOTOMETRY OF NUCLEAR-DNA IN BUDS AND FLOWERS OF NICOTIANA-TABACUM

Feulgen/DNA absorptions were measured by scanning cytophotometry on the nuclei of meristematic cells in buds and flowers of Nicotiana tabacum developing in vivo or regenerating in vitro from thin cell layers excised from different plant parts and characterized by significant changes in the amount and organization of nuclear DNA. The results obtained indicated that, in vivo, nuclear DNA contents were significantly higher in flowers than in buds. Significant differences in the amount of nuclear DNA were also observed when comparing buds developing in vivo and in vitro and, among the latter, those regenerated from explants taken from different plant parts. Flowers developed in vivo and those regenerated in vitro, independent of which explants they were formed from, always have the same DNA content. These findings seem to suggest that rapid quantitative changes in the nuclear DNA having a possible role in developmental regulation can occur in Nicotiana tabacum

RNA-SYNTHESIS IN THE EMBRYO SUSPENSOR OF PHASEOLUS-COCCINEUS AT 2 STAGES OF EMBRYOGENESIS, AND THE EFFECT OF SUPPLIED GIBBERELLIC-ACID

RNA synthesis in giant cells containing polytene chromosomes in the embryo suspensor of Phaseolus coccineus was analyzed by autoradiography after [ 3H]-uridine treatment. Embryos at the heart-shaped stage of development and at a cotyledonary stage were studied. Discontinuous labelling of the polytene chromosomes was always observed. The chromosomes were subdivided into segments (chromosome regions) which behaved as functional units, since discontinuous labelling was never seen within any of the regions. It was found that most chromosome regions were engaged in RNA synthesis to different degrees at the two embryo developmental stages. Regions showing identical labelling patterns tended to lie close together in the chromosome arms and to keep their functional activity coordinated at both stages of embryo development. The chromosome regions bearing 18 S+25 S ribosomal genes were never simultaneously active in RNA synthesis and different regions were preferentially transcribed at each stage of embryo development. However, at both stages, all the chromosome regions bearing 5 S ribosomal genes showed comparable labelling frequencies. The effect on transcription of gibberellic acid (GA 3) treatments was also studied. At both embryo developmental stages, GA 3 enhanced the rate of RNA synthesis in the polytene suspensor cells. The frequency with which certain chromosome regions were transcribed was also increased significantly (P≤0.001) and this stimulatory effect was greater in embryos at the cotyledonary stage than in heart-shaped embryos. At the latter developmental stage, RNA synthesis was repressed by GA 3 in a few chromosome regions. These results are discussed briefly in relation to previous findings using different methods of studying the organization of polytene chromosomes and the functional activity of the embryo suspensor of Phaseolus coccineus

CYTOLOGICAL LOCALIZATION OF FAST RENATURING AND SATELLITE DNA-SEQUENCES IN VICIA-FABA

DNA sequences reassociating within a Cot value of 1.8×10 -1 and those producing a light satellite in a CsCl density gradient were isolated from Vicia faba DNA and hybridized in situ on squashes of roots of the same species. Silver grains were seen to be scattered over both the interphase nuclei and the metaphase chromosomes after hybridization with fast renaturing DNA sequences, indicating these are fairly regularly interspersed in the V. faba genome. Clustered labeling occurred after hybridization with satellite DNA sequences, indicating these are clustered in the genome. The localization of satellite DNA in chromosomes appeared to correspond closely to the position of the bright bands detectable after staining with quinacrine mustard. After hybridization with both DNA probes, labeling intensity over the nuclei of meristematic cells was higher than that over the nuclei of differentiating and/or differentiated cells. These results are discussed in relation to the structure of the cell nucleus, the mechanism of quinacrine banding and to previous data suggesting underrepresentation of nuclear repeated DNA sequences in differentiating V. faba root cells