Cytochemical and Autoradiographic Analyses on the Embryo Suspensor Cells of<i>Phaseolus Coccineos</i>

Abstract

SUMMARYSpecific staining methods (Feulgen, methyl green-pyronin, toluidine blue molybdate, acridine orange), digestion tests (DNase and RNase) and autoradiographic techniques (incorporation of tritiated thymidine, uridine, lysine, tryptophan; DNA detection by means of tritiated actinomycin D) have been used to study the embryo suspensor of Phaseolus coccineus. Most of material studied consisted of developing seeds in which the cotyledons of the embryo occupied one third to one half of the endospermatic cavity. In the typically club-shaped suspensor, both the « handle » portion (which contains cells with low to medium degree of endopolyploidy) and the « knob » portion (which contains polytene chromosome cells) have been analyzed.Indications have been found that the cells with low and medium degree of endopolyploidy undergo extra DNA synthesis (gene amplification) on many heterochromatic chromosome regions during early stages of embryogenesis. Later on, the extra DNA synthesized seems to be released, from the chromosomes into the nucleoplasm and from here into the cytoplasm, as micronucleoli or clusters of micronucleoli. The micronucleoli are formed by a spherical mass of ribonucleoproteins covered by a layer of DNA, sometimes at least, traversed by thin DNA strands. In addition to extrusion into the cytoplasm of micronucleoli, the nuclei of the « handle » portion of the suspensor are active in the extrusion of nuclear material in form of « blebs »: whether these contain only ribonucleoprotein or also bear DNA in a condition different from that of the micronucleoli cannot be decided. At the developmental stages studied, the cells of the « handle » portion of the suspensor do not practically incorporate thymidine and only rarely incorporate lysin (in 2 out of 11 suspensors studied); but they are active in the incorporation of tryptophan and uridine. After 30 minutes of feeding with 3H-uridine, labelling in the suspensor nuclei is restricted to the nucleoli, whilst in the embryo cells (both plumule and cotyledons), the nuclei (chromatin) are heavily labeled, but the nucleoli are practically unlabeled.In the polytene chromosome cells of the suspensor (« knob » portion) the following observations have been made: i) Essential characteristics of polytene chromosomes. Several aspects of the structural organization of nucleolar puffs and other puffs, of the lampbrush state of chromosomes or parts thereof, of the chromosome endoreduplication stage are described. In a few cells, all chromosomes are surrounded by an RNA (ribonucleoprotein) sheath; in others, only some chromosomes and parts of them bear the RNA sheath; in othe (the majority) this RNA sheat is absent;ii) Nucleolar apparatus. It is formed by the main nucleoli organized by the two pairs of SAT-chromosomes (I and V) and by the micronucleoli produced in connection with extra DNA replication in many heterochromatic regions of the chromosome complement. In the main nucleoli, the intranucleolar chromatin is made by two distinct parts: the « nucleolar puff », formed by the more or less extended strands of the chromosomal regions involved in nucleolus, organization, and the «e xtra chromosomal chromatin », which is detached from the chromosomes following extra DNA replication in the organizers and satellites. It is suggested that, in Phaseolus nucleoli, the matrix is due to synthetic activity of the nucleolar puff and the vacuoles are made by the ribonucleoproteins synthetized by the extra DNA. A few cells are found to contain a large « lateral » nucleolus, organized, in an intercalary region of a polytene chromosome, by a part of the strands forming the chromosome.iii) Labeling behaviour. Apart from a few cases of polytene nuclei caught during the process of chromosome endoreduplication, incorporation of thymidine in polytene chromosomes occurs on heterochromatic regions only; these regions also incorporate lysine (thus, the extra DNA is complexed with histones) and uridine: this last compound also gives the typical incorporation pattern in nucleoli, euchromatin and cytoplasm. Incorporation of lysine and tryptophan, in both, nucleus and cytoplasm, is very active.iv) Functiónal asynchrony within a chromosome region. In addition to cases of formation of a « lateral » nucleolus (see above), cases are described in which, within a band, some strands show a structure or a behaviour (incorporation of precursors, extra DNA synthesis) different from other strands.v) Physiological role of the suspensor. The development of a haustorial membrane system and the several aspects of functional activity shown by the suspensor cells indicate that the suspensor may be engaged in the production of molecules to be transferred to the embryo. The possibility is also considered that a division of labor between embryo cells and suspensor cells might occur during developmentt.The phenomena observed in P. coccineus find a striking parallel in the phenomena described in the polytene chromosome cells of Sarcophaga, Hybosciara and, occasionally, in Chironomus.