Fine Structure and Staining Behaviour of Heterochromatic Segments in Two Plants

0 John Innes Institute , Colney Lane, Norivich, England 1 Present address: School of Biological Sciences, University of East Anglia , Norwich, England With the use of the light and electron microscopes, the chromosomes of Fritillaria lanceolata and Scilla sibirica are shown to differ in respect of the heterochromatin they contain. In root meristems of the former, the heterochromatic regions (H-segments) were recognizable at all phases of the mitotic cycle by their slighter opacity to electrons than that of euchromatic parts. This was due both to less tight packing of the chromatin fibrils and lower opacity of the fibrils themselves, even though both had the same diameter, about 3 nm. - After suitable cold treatment, the somatic chromosomes of certain plant species show segments of constant length and position which at metaphase and anaphase stain less intensely with the Feulgen reaction and basic dyes than the rest of the chromosome (Darlington & La Cour, 1938, 1940, 1941; Geitler, 1940; Haga, 1944; Haga & Kurabayshi, 1953; La Cour, 1951; Dyer, 1963, and others). A similar finding was obtained by Callan (1942) in mitoses from 3 species of newt. Darlington & La Cour found that these regions corresponded to the condensed heterochromatic regions (H-segments) seen in interphase nuclei, first intensively studied and named by Heitz (1929, 1932). Darlington & La Cour suggested that, in chilled mitoses, the differential reactivity of the H-segments was due to competition between these regions and the euchromatic parts for a reduced nucleic acid supply. This view was challenged by Wilson & Boothroyd (1941) who considered that the appearance of the H-segments was due to a differential contraction in these regions and the euchromatic parts. It was deduced that, from measurements in chromosomes of Trillium erectum, the H-segments were less contracted than the euchromatic parts, when compared with unchilled chromosomes. Bailey (1949) also reached this conclusion. Attempts to resolve this problem by Feulgen photometry have produced conflicting results. Evans (1956), La Cour, Deeley & Chayen (1956) and Heyes & Shaw (1958) all obtained results which suggested that there was less DNA in the nuclei of cold-treated plants, whereas those obtained by Woodard & Swift (1964) did not. Heyes & Shaw were not able to confirm their results by chemical analysis, and indeed found more DNA per cell in the chilled plants. The premise that 'nucleic acid starvation' is responsible for the differential reactivity of the H-segments has received little or no support from autoradiography, in that clearly DNA synthesis is not involved (La Cour, i960; Boothroyd & Limade-Faria, 1964; Woodard & Swift, 1964). Haque (1963) found labelled euchromatic regions and unlabelled H-segments in chromosomes of Trillium grown 5 days at 1 C after 16-h treatment with [3H]thymidine and 24 h of further growth at 22 C. He suggested the possibility that this situation could arise by actual loss of preformed DNA in the latter segments. Asynchrony of DNA replication in the 2 regions might, however, provide an explanation for Haque's findings. The present study was undertaken with the view that a comparative study of the fine structure of euchromatic and heterochromatic regions in nuclei of meristematic cells of plants grown at both relatively warm and low temperature might provide an explanation for the differential reactivity of H-segments in chilled chromosomes. Fritillaria lanceolata was chosen for this purpose, because all but 2 of its 24 chromosomes have relatively large H-segments which are mostly situated close to the centromeres (La Cour, 1951). A similar comparison was also made in nuclei of meristematic cells of Scilla sibirica (after growth at normal temperatures), a species in which with chilling, the H-segments are not revealed at metaphase (La Cour, 1951). In England, Fritillaria lanceolata is usually grown in pots in a cold frame or unheated glasshouse and roots are produced between late October and March. In order to avoid growth in cold spells, 2 control plants were kept in a heated glasshouse at 18-20 C for 14 and 28 days, respectively. The cold-treated plants were chilled at 2 CC for 4 days. Scilla sibirica was grown in an unheated glasshouse. Root tips were fixed in 10% formalin in phosphate buffer (pH 76) plus o-i % calcium chloride and 0-3% sucrose for 18-24 h. After fixation the specimens were rinsed briefly in water and then transferred to Caulfield's osmium-sucrose (plus calcium chloride as above) for 2 h to stabilize the fixation image and enhance its contrast in the electron microscope. To obtain further contrast they were stained 1 h in 2 % uranyl acetate in 70 % acetone as a step during dehydration. Epon was used for embedding. Thick (0-5 /(in), relatively thin, and ultrathin sections were cut with a Cambridge-Huxley microtome. The intermediate class, in the range of 65-76 ran, were used for the study of identical sections in sequence with the light and electron microscopes, using the technique perfected by Wells & La Cour (1971). These and the ultrathin sections were subsequently stained in lead acetate (Millonig, 1961); the former after observation in the light microscope. The thick sections, required for the light microscope only, were stained in hot toluidine blue, as in the method employed for sequential studies. The micrographs were taken with an Elmiskop 1 a and all measurements made after calibrated magnification, using the catalase method of Wrigley (1968). The heterochromatin of F. lanceolata The H-segments in chromosomes of F. lanceolata fuse at telophase to form a variable number of chromocentres which in interphase nuclei stain intensely with Feulgen and basic dyes when root tips are processed as squashes (Fig. 4). The staining behaviour of the chromocentres in such preparations was not noticeably different in nuclei from root tips of plants grown at 18-20 C or at 2 C for 4 days. Metaphase plates from plants grown at these 2 temperatures are illustrated in Figs. 5 and 6, respectively. The H-segments are clearly visible in the metaphase from the coldtreated plant, where it can also be seen that the chromosomes are obviously supercontracted. Microphotographs and micrographs taken in sequence with the light and electron microscopes of the same sections of prophase and anaphase chromosomes from root tips of a chilled plant are shown in Figs. 7-10. It will be apparent that the markedly paler staining of the H-segments with toluidine blue is closely paralleled by the low contrast of their image with electrons. When metaphases and anaphases were examined with the electron microscope in thin sections of root tips from a control plant kept for 14 days at 18-20 C, we were surprised to find that the H-segments were invariably as clearly defined as in sections from chilled material. Because of the remote possibility that the differential reactivity of the H-segments was due to an incipien (...truncated)