Aluminum effects on embryo suspensor polytene chromosomes of Phaseolus coccineus L

Aluminum (Al) represents a widespread environmental pollutant, with severe toxic impacts on plants. In this study, we documented for the first time the structural and functional responses induced by two concentrations of AlCl₃ (10^?2 M and 10^?1 M) in the polytene chromosomes that characterize the chromatin organization in the embryo suspensor cells of Phaseolus coccineus. Polytene chromosomes showed signs of dose-dependent genotoxicity following AlCl₃ treatments with a significant increase in both chromatin stickiness and chromatin fragmentation. Polytene chromosomes specifically reacted to AlCl₃ also in terms of DNA and RNA puffing activity: with respect to the control, the treatments promoted ex-novo and/or inhibited puff formation along chromosome arms, suggesting a fine modulation of the differential genome activity in response to the treatments. The nuclei of suspensors from control and treated seeds showed nucleoli mainly arranged by more than one NOR-bearing chromosome. In addition, AlCl₃ treatments affected the frequency of nucleoli organized by singular organizer chromosomes, with an increase in the frequencies of nucleoli organized by chromosome II and a reduction in the frequencies of those organized by chromosomes I or V. These results confirm that, also in our system, nucleolus may react as stress response organelle.     

Identification of gibberellin A1 in the embryo suspensor of Phaseolus coccineus

By combined gas chromatography-mass spectrometry the gibberellin present in suspensors of heart-shaped embryos of Phaseolus coccineus has been identified as Gibberellin A₁ (GA₁). The amount of GA₁ in 2000 suspensors (452 mg), as estimated by gas chromatography. was 4g. The presence of GA₁ in suspensors of P. coccineus is discussed in relation to our present knowledge of the occurrence of many gibberellins in developing seeds and immature fruits of the same species.Abbreviations FID flame ionization detector - GA gibberellin - GC gas chromatography - MS mass spectrometry - PGC preparative gas chromatography - Stage A heart-shaped embryo - Stage B cotytedonary embryo - TMS trimethylsilyl     

Cytological localization of ribosomal cistrons in polytene chromosomes of Phaseolus coccineus

Tritiated ribosomal RNA (rRNA) was prepared from hypocotyls of Phaseolus coccineus grown in liquid culture in the dark and in presence of 5-³H-uridine. A mixture of the 18S and 25S ³H-rRNA fractions was used for hybridization with DNA in the polytene chromosome cells of the embryo suspensor of P. coccineus. It was shown that the ribosomal cistrons (rDNA) are located in the nucleolus organizing system (satellite, nucleolar constriction and organizer) of the satellited chromosome pairs I (S₁) and V (S₂), in the proximal heterochromatic segment of the long arm of chromosomes S₁ and in the terminal heterochromatic segment of chromosome pair II. The micronucleoli which are produced by the satellite and nucleolus organizer of the chromosome pair S₁ contain rDNA; on the contrary, no rRNA-DNA hybridization is found in the DNA containing granules which are produced by the satellite and nucleolus organizer of chromosome pair S₂. The DNA which is amplified during production of DNA puffs at some chromosomal regions apparently does not code for ribosomal RNA (no detectable rRNA-DNA hybridization).Publication no. 62 from the Laboratorio di Mutagenesi e Differenziamento, Consiglio Nazionale delle Ricerche, Pisa. Part of the investigation was supported by Contract SC 001/076-69-1 BIAN between the European Atomic Energy Community and the University of Pisa, Institute of Genetics.     

Gibberellins in suspensor,embryo and integument from very young seeds of Phaseolus coccineus L.

Endogenous gibberellins (GAs) were extracted from suspensor, embryo and integument of very young seeds of Phaseolus coccineus L. and detected by combined gas chromatography-mass spectrometry (GC-MS). Results show the presence of one C₂₀-GA, GA₄₄ and five C₁₉-GAs in the suspensor: GA₁, GA₄, GA₅, GA₆ and GA₈, and four C₁₉-GAs in the integument: GA₁, GA₅, GA₆ and GA₈. Only traces of GA₁ and GA₅ were identified in the embryo. A compound structurally related to GAs was identified as tetrahydroxy-Kauranoic acid in suspensor, integument and, only in trace amounts, in the embryo.     

Structure and replication of Phaseolus polytene chromosomes

The normal morphology of the polytene chromosomes of the embryo suspensor of Phaseolus coccineus is that of a tightly condensed cord with heavily Feulgen staining centromeric heterochromatic regions (α-heterochromatin) and other accessory heterochromatic regions (β-heterochromatin). The replication pattern of the chromosomes has been determined by autoradiographic analysis of material pulsed with ³H-thymidine for various lengths of time. The DNA replication cycle reqires 4–6 hours for completion. During replication chromosome structure becomes diffuse and the β-heterochromatic regions are indistinguishable from the euchromatic regions. The euchromatin is the first to replicate, and replication begins simultaneously at numerous sites in the euchromatin. The β-heterochromatin replicates next, and finally the centromeric heterochromatin. Replication is essentially complete in each of these parts of the chromosome before DNA synthesis begins in the next. The chromosomes are composed of numerous longitudinally running Feulgen positive strands, the equivalent portions of which replicate simultaneously. This indicates that there must be close control of the replication cycle in sister strands.     

Callus formation from the suspensor ofPhaseolus coccineus in hormone-free medium: a cytological and DNA cytophotometric study

Summary Intact embryos (with suspensor) ofPhaseolus coccineus (2 n=2 x=22) in different stages of development were grownin vitro on hormone-free medium under conditions favoring callus formation from the portion of the suspensor proximal to the embryo (handle portion). It was shown that callus formation was dependent upon the developmental stage of the embryo and the integrity of the embryo-suspensor system.Callus formation from the handle portion of the suspensor, whose cells have nuclei with DNA values from 4 C to 128 C, was initiated by amitosis (nuclear fragmentation) which led to binucleate and multinucleate cells. Amitosis was followed by mitosis of both nuclei resulting from a previous amitosis and nuclei of euploid cells (mostly diploid) pre-existing in the suspensor. The majority of mitoses (88%) had either a reduced chromosome number (hypodiploid, haploid, and hypohaploid) or the diploid number (22 chromosomes).The very high incidence of cells with reduced chromosome number in the suspensor callus is discussed in relation to its mechanism of origin and to its possible exploitation in plant regeneration experiments.     

Pattern of binding of tritiated actinomycin D to Phaseolus coccineus polytene chromosomes. II. The entire chromosome complement

Abstract

The pattern of labelling by tritiated actinomycin D (³H-AMD) on polytene chromosomes in the embryo suspensor cells of Phaseolus coccineus is described in preparations exposed for different times to photographic emulsion. It is observed that: a) each chromosome pair displays a typical labelling pattern; b) heterochromatin close to the centromere does not label in the same way in different chromosome pairs; c) in some chromosome pairs, different bundles of strands in one an the same chromosome segment display differences in ³H-AMD labelling. These results allow an improved characterization of the Phaseolus polytene chromosomes and are discussed in relation to the possible factors determining the differential binding of ³H-AMD to the chromatin.     

Detection of rRNA and phaseolin genes on polytene chromosomes of Phaseolus coccineus by fluorescence in situ hybridization after pepsin pretreatment.

This is the first report of fluorescence in situ hybridization (FISH) on plant polytene chromosomes. Different protease pretreatments have been tested to improve fluorescence in situ hybridization FISH on polytene chromosomes of a plant, Phaseolus coccineus, with the aim to enable the detection of low-copy genes. The structural preservation of the chromosomes and the distinctness of the FISH signals were comparatively analysed with a probe for the ribosomal RNA genes after digestion with pepsin and trypsin. The pepsin pretreatment resulted in a general loosening of chromatin with good conservation of chromosome morphology and an increased number and density of signal points. The six nucleolus organizers exhibited significant differences in condensation. The pretreatment with pepsin enabled the detection of the low-copy genes encoding the seed storage protein phaseolin.     

Localization of phaseolin genes in the polytene chromosomes ofPhaseolus coccineus (Leguminosae)

The location of the genes for the major storage protein, phaseolin, was determined in the polytene chromosomes of the embryo-suspensor ofPhaseolus coccineus, cv. Hammond's Dwarf Scarlett, byin situ hybridization with a³⁵S-labelled cloned probe. The genes were adjoined to chromosome pair no. 3. The distribution of the label indicates a puffed state of the sites during embryogenesis of the legume studied. The location of the phaseolin genes was certified by hybridization of a ribosomal DNA probe to the polytene chromosomes, because their position is well known.     

Distribution of 5-methylcytosine-rich regions in the polytene chromosomes of Phaseolus coccineus embryo suspensor as shown by the immunoperoxidase technique

5-Methylcytosine (5-mC) has been visualized in polytene chromosomes of Phaseolus coccineus, scarlet bean using specific antibodies to 5-mC and the immunoperoxidase technique. The results obtained indicate that most heterochromatic regions are methylated, even though the frequency of methylation is highly variable and sometimes low. A preferential binding of anti-5-mC to centromeric heterochromatic blocks was observed. Comparison between anti-5-mC binding and the results of hybridization with highly repetitive DNA and satellite DNA shows, moreover, that centrometric heterochromatic regions hybridize in particular with both DNAs. This finding is consistent with the fact that repetitive DNA and satellite DNA are methylated to a considerably greater extent than main band DNA, in line with many data to be found in the literature. The binding pattern of anti-5-mC that we observed also suggests that methylation does not occur in all classes of repetitive DNA. The high variability of band methylation frequency is discussed in relation to a possible characteristic DNA composition of the band.     

The effect of the suspensor and gibberellic acid on Phaseolus vulgaris embryo protein synthesis

The role of the suspensor in the early development of the dicot embryo has been described as merely an anchor or, conversely, as the major route of nutrients into the embryo. In order to further elucidate the role of the suspensor we have examined protein synthesis in early 0.2-mm and late heart stage 0.5-mm Phaseolus vulgaris (var. Taylor's Horticultural) embryos in tissue culture. Protein synthesis was examined in embryos and suspensors. Our results showed that in 0.2-mm embryos virtually all protein synthesis was dependent on an attached suspensor. Maximum protein synthesis in 0.5-mm embryos was observed when embryos were cultured attached to the suspensor. The levels were moderately decreased when the embryo was cultured detached from or without the suspensor. Gibberellic acid at 10(-6) to 10(-7) M elicited the same protein diversity and greater [35S]methionine incorporation than did the attached suspensor in 0.2-mm embryos. Embryos of 0.5 mm did not appear to be differentially responsive to various gibberellin concentrations.     

The effect of the suspensor and gibberellic acid on Phaseolus vulgaris embryo protein content

The role of the suspensor in the early development of the dicot embryo has not yet been defined. It has been described as merely an anchor and also as the major route of nutrients into the embryo. In order to further elucidate the role of the suspensor, early 0.2-mm and late heart stage 0.5-mm Phaseolus vulgaris (var. Taylor's Horticultural) embryos have been examined in tissue culture. It is known that Phaseolus embryos in culture at low osmotic potential will germinate precociously and that embryos in culture at high osmotic potential will either fail to grow or form callus. Optimum sucrose concentrations for continued, normal embryonic development of 0.2 mm and 0.5 mm P. vulgaris in tissue culture with Gamborg B5 medium were determined to be 12 and 6%, respectively. Protein content was examined in embryos and suspensors. Results showed that both 0.2- and 0.5-mm embryos required an attached suspensor for maximum protein content. Protein levels were substantially decreased when the embryo was cultured detached from or without the suspensor. Gibberellic acid at 10(-6) to 10(-7) M restored the protein content to that of freshly excised embryos.     

Detection and localization onDrosophila melanogaster polytene chromosomes of sequences homologous to oncogeneyes

Sequences homologous to oncogeneyes (Y73/Esh/sarcoma viral oncogene cDNA) in theDrosophila melanogaster Oregon genome were detected byin situ hybridization on salivary gland chromosomes. Three separate sites, 8D/X, 57BC/2R and 95CD/3R, were identified. Presence of sequences highly homologous toyes in the genomic DNA was confirmed by dot blot hybridization under high stringency conditions.     

Correlation of structure and RNA synthesis in the nucleolus-organizing polytene chromosomes of Phaseolus vulgaris

The relationship between RNA synthesis and morphology of the nucleolus-organizing polytene chromosomes in the highly endopolyploid suspensor cells of Phaseolus vulgaris has been studied by actinomycin D treatment, temperature lowering, and H³-uridine autoradiography. Actinomycin D and low temperature induce a condensation of the giant chromosomes, particularly of the nuoleolus organizers and of the intranucleolar regions of the chromosomes. RNA synthesis occurs in the extended state of the chromosomes, but it ceases in the highly condensed state caused by the treatment of the cells either with actinomycin D or with low temperature.     

Cytological localization of the genes for the four classes of ribosomal RNA (25S, 18S, 5.8S and 5S) in polytene chromosomes of Phaseolus coccineus

Homologous tritiated 25S, 18S and 5.8S rRNAs were used separately for in situ hybridization to the polytene chromosomes of the embryo suspensor cells of Phaseolus coccineus. Hybridization occurred at the same chromosomal sites which were labeled in previous in situ hybridization experiments with 25+18S rRNAs in the same material (Avanzi et al., 1972), namely: nucleolus organizing system (satellite, nucleolar constriction and organizer) of chromosome pairs I (S₁) and V (S₂), proximal heterochromatic segment of the long arm of chromosome pair I, and terminal heterochromatic segment of chromosome pair II. Competition hybridization experiments confirmed for P. coccineus the high sequence homology between 25S and 18S rRNA already known for other plants.Homologous ¹²⁵I-5S rRNA was found to hybridize to three sites in the polytene chromosomes of P. cocdneus: the proximal heterochromatic segment in the long arm of chromosome pair I (which also bears the sequences complementary to 25S, 18S and 5.8S RNAs), most of the proximal heterochromatic segment plus a small portion of adjoining euchromatin in the long arm of chromosome pair VI and the large intercalary heterochromatic segment in the same chromosome pair. Simultaneous labeling of the two 5S RNA sites in chromosome VI was quite rare (3%), the rule being labelling of one site to the exclusion of the other, with a labeling frequency of 43.7% and 53.3% for sites no. 1 and no. 2 respectively. These results are interpreted as being due to differential hybridizability of chromosomal sites such as described in other materials.