Giemsa C-banding of Rhoeo (Commelinaceae)

The C-banded karyotypes of the three known varieties of Rhoeo are presented for the first time. Each variety displayed the same banding pattern with C-bands identified at all centromeres and some telomeres. In addition, the karyotype of a rare bivalent-forming Rhoeo was ascertained to consist of two complexes using C-banding. The nature and location of Rhoeo's C-bands are discussed.This paper represents part of a dissertation submitted for a Ph.D. thesis at West Virginia University.     

Control of Ribonuclease and Acid Phosphatase by Auxin and Abscisic Acid during Senescence of Rhoeo Leaf Sections

We report the effects of abscisic acid and auxin (α-naphthalene acetic acid) on regulation of enzyme synthesis during senescence of leaf sections of Rhoeo discolor Hance. Abscisic acid always accelerates the onset of and enhances the magnitude of the increase in activity of acid phosphatase; this is followed by an acceleration of the onset of a rapid increase in free space.     

Transitory increase in chromosomal DNA (Fleulgen) during floral differentiation in Rhoeo discolor.

Scanning cytophotometric measurements on 3200 telophase and 1700 interphase nuclei (Feulgen-stained) in vegetative and reproductive buds of Rhoeo discolor revealed a transitory increase in staining intensity in more than half of the cells in early differentiating floral buds. The differences between the vegetative and floral nuclei are significant at the 0.001 level of probability and highly reproducible, independent of the type of hydrolysis used. We suggest that the different Feulgen extinction values reflect different nuclear DNA amounts, because methodical errors can fairly be excluded. The occurrence of an extra DNA (control DNA) of the kind of the floral DNA' detected by Wardell and Skoog (1973) and Wardell (1976) in tobacco is discussed.     

Plasmolytische Beobachtungen an den Epidermiszellen von Rhoeo discolor

Zusammenfassung Die nachfolgenden Beobachtungen beziehen sich auf das klassische Objekt für grenzplasmolytische Bestimmungen, die Epidermiszellen der Blattunterseite vonRhoeo discolor.Die Schnitte wurden in der von Fitting (1915) angegebenen Weise hergestellt, nur mit der Abweichung, daß die Mittelrippe nicht halbiert wurde. Die Schnittränder, in deren Nähe sich die untersuchten Zellen befinden, verlaufen somit senkrecht gegen die Längsrichtung des Blattes. Allein die Epidermiszellen der eigentlichen Mittelrippe wurden beobachtet. Die Schnitte hatten eine Dicke von etwa 0,2–0,4 mm und. enthielten somit außer der Epidermis mehrere Zellschichten des subepidermalen Gewebes. Zur Herstellung der Lösungen wurde immer Helsingforser Leitungswasser (kalkarmes, chemisch gereinigtes Flußwasser von fast neutraler Reaktion) verwendet.     

Ribosomal DNA,tri- and bi-partite pericentromeres in the permanent translocation heterozygote <Emphasis Type="Italic">Rhoeo spathacea</Emphasis>

High- and low-stringency FISH and base-specific fluorescence were performed on the permanent translocation heterozygote Rhoeo spathacea (2n = 12). Our results indicate that 45S rDNA arrays, rDNA-related sequences and other GC-rich DNA fraction(s) are located within the pericentromeric regions of all twelve chromosomes, usually colocalizing with the chromomycin A₃-positive bands. Homogenization of the pericentromeric regions appears to result from the concerted spread of GC-rich sequences, with differential amplification likely. We found new 5S rDNA patterns, which suggest a variability in the breakpoints and in the consequent chromosome reorganizations. It was found that the large 5S rDNA locus residing on each of the 8E and 9E arms consisted of two smaller loci. On each of the two chromosome arms 3b and 4b, in addition to the major subtelomeric 5S rDNA locus, a new minor locus was found interstitially about 40% along the arm length. The arrangement of cytotogenetic landmarks and chromosome arm measurements are discussed with regard to genome repatterning in Rhoeo.     

Chromosome distribution and catenation in Rhoeo spathacea var. concolor

The twelve chromosomes of Rhoeo spathacea variety concolor are arranged in a definite sequence in a ring at meiosis. Identification of all the 12 chromosomes was possible in 119 diakinesis and metaphase I cells. — Pollen viability was measured to be 36.54% by cotton blue staining procedure. Forty five of 56 metaphase I cells (80.36%) had adjacent distribution. Each of the 12 chromosomes was equally likely to be involved in adjacent distribution regardless of their sizes and heterobrachialness. Adjacent distribution occurred randomly at each arm-position in the ring regardless of the lengths of the arm-pairs. — The most frequent chromosome configuration at diakinesis and metaphase I was a chain-of-12 chromosomes (41.18%). Cells with 1 to 4 chains of chromosomes were observed. The observed frequencies of various configurations were in good agreement with the calculated frequencies. The mean number of chiasmata was 10.90 per cell and 0.908 per pair of chromosome arms. The 131 chiasma failures were distributed at random among the 12 arm-positions. Since the lengths of arm-pairs in the ring vary, the randomness may mean that chiasma formation was limited to short terminal segments on all chromosomes.     

The relation between synapsis and chiasma formation in Rhoeo spathacea

Rhoeo spathacea (Schwartz) Stearn (2n=12) is a complete translocation heterozygote in which a ring of 12 chromosomes is often observed from diakinesis through metaphase I. A new hypotonie bursting technique on primary microsporocytes made possible visualization of the complete pattern of pachytene synapsis in R. spathacea by light and electron microscopy. Analysis showed that most of the length of a chromosome never synapsed and that relational twisting of the lateral elements was prominent in the distal segments of most synaptonemal complexes (SCs). The most common situation was 12 relatively short SCs representing the 24 synapsed terminal segments of the 12 chromosomes; 11 and 10 SCs were also observed but less frequently, indicating one and two synaptic failures, respectively. Similarly, in diakinesis a ring of 12 chromosomes was most common, but a chain of 12 chromosomes and two chains of chromosomes could also occur. These situations indicated zero, one, and two chiasma failures, respectively. The frequencies of synaptic failure (0.021) and of chiasma failure (0.022) were essentially the same as were the distributions of cells with different numbers of synaptic and chiasma failures. These comparisons indicate that if synapsis occurs between two distal segments of the chromosomes of R. spathacea, a chiasma will almost certainly follow. A diagram to illustrate the arrangement of mid-pachytene chromosomes and a model to explain the origin of relational twisting of lateral elements in SCs are presented.     

Temperature and chiasma formation in Rhoeo spathacea var. variegata

A clone of Rhoeo spathacea var. variegata was exposed to a constant temperature of 37°C to examine the effects of high temperature on the chiasma frequency. Rhoeo was chosen for this study for its unusual meiotic chromosome configurations (rings and chains) at diakinesis and the first metaphase which permitted the calculation of terminal-chiasma frequency, and for its genetic homogeneity among plants in a clone. Plants treated for 12 h and 24 h at 37°C had similar mean terminal-chiasma frequencies to that of controls (10.93±0.11, 10.95±0.09 and 10.64±0.07 respectively), while those treated for 36 h, 48 h or 60 h had lower mean terminal-chiasma frequencies than that of controls (8.06±0.18, 4.25±0.15, 0.76±0.07 and 10.64±0.07 respectively). Analysis of variance was performed to find out whether the mean terminal-chiasma frequencies resulted from different treatments were significantly different from one another. The results of the analysis indicated that prolonged exposures to high temperature significantly reduced the chiasma frequencies.     

Isolation of Highly Acylated Anthocyanins from Commelinaceae Plants,Zebrina pendula,Rhoeo spathacea and Setcreasea purpurea

Acylated anthocyanins in the Commelinaceae were distinguished from other substances by high-performance liquid chromatography (HPLC) with concurrent three-dimensional photodiode- array detection (250~700nm), and four new acylated anthocyanins were isolated by preparative HPLC.

According to the fast atom bombardment mass spectral (FABMS) analysis, alkaline- hydrolysis and the absorbance ratio between λ_max of around 320 nm and that around 500 nm, the new pigments were characterized as follows: zebrinin [MW (as flavylium ion) 1553, caffeic acid × 4] from Zebrina pendula; monodecaffeylzebrinin [MW 1391, caffeic acid × 3] from Zebrina pendula; rhoeonin [MW 1433, ferulic acid × 3] from Rhoeo spathacea’, and setcreasin [MW 1609, ferulic acid × 4] from Setcreasea purpurea.     

The asymmetric meiosis in pentaploid dogroses (Rosa sect. Caninae) is associated with a skewed distribution of rRNA gene families in the gametes

In pentaploid dogroses, Rosa section Caninae (2n=5x=35), the pollen transmits one basic genome (x=7) derived from the seven segregating bivalents, whereas the egg transmits four basic genomes (4x=28) one set derived from the segregation of seven bivalents and three sets of univalent-forming chromosomes. Chromosomes from all five genomes carry 18-5.8-26S nuclear ribosomal DNA (rDNA) sites. This mode of sexual reproduction, known as permanent odd polyploidy, can potentially lead to the independent evolution of rDNA on bivalent- and univalent-forming chromosomes. To test this hypothesis, we analyzed rRNA gene families in pollen and somatic leaf tissue of R. canina, R. rubiginosa and R. dumalis. Six major rRNA gene families (alpha, beta, beta' gamma, delta and epsilon) were identified based on several highly polymorphic sites in the internal transcribed spacers (ITSs). At least two of the major rRNA gene families were found in each species indicating that rDNAs have not been homogenized across subgenomes. A comparison of ITS1 sequences from leaf and pollen showed differences: the shared beta rRNA gene family was more abundant among pollen clones compared to leaf clones and must constitute a major part of the rDNA loci on bivalent-forming chromosomes. The gamma and delta families were underrepresented in pollen genomes and are probably located predominantly (or solely) on the univalents. The results support the hypothesis that pentaploid dogroses inherited a bivalent-forming genome from a common proto-canina ancestor, a likely donor of the beta rDNA family. Allopolyploidy with distantly related species is likely to have driven evolution of Rosa section Caninae.     

Mechanismus der nondisjunktionalen Chromosomenverteilung und die Ursachen der Pollensterilität bei Rhoeo spathacea

Different types of chromosome configurations during the meiotic metaphase I of pollen mother cells of Rhoeo spathacea were identified. The rates of these configurational types show only little variation under extreme environmental conditions. For nearly all configurational types the ratio of chromosome rings to chains was found to be the same. — During metaphase I the chromosomes arrange in the following way: At the beginning, due to spatial properties of the pollen mother cells, spindle fibers are attached to the centromeres of only a few chromosomes. Starting with these firstly attached ones all chromosomes are arranged in the equatorial region, accomplished by a contraction of the chromosomal apparatus. Frequently this results in the nondisjunctional distribution of two pairs of chromosomes. The theoretical calculations of the frequencies of the various configurational types to be expected in this observed mechanism were in agreement with the actually observed frequencies. — The arrangement of chromosomes during late metaphase I and their actual distribution in anaphase I show that about one half of the unequal types have reorientated to equal ones. — The number of pollen grains and the percentage of sterility depend extensively upon environmental conditions. When the conditions are more favourable, more pollen grains are produced and the degree of sterility is lowered. — The decay of pollen occurs during several stages: In the first stage, lasting from the dissolution of the layer enveloping the tetrad until the end of pollen mitosis I, approximately 55 per cent of the pollen grains decay due to the haploid lethal factor and the deficiencies of the (n-l)-chromosome sets. In a second stage, from the end of pollen mitosis I to anthesis, up to additional 30 per cent of the grains decay. This is largely determined by environmental factors, possibly acting upon the periplasmodium, and disharmonic combinations of chromosomes. All grains with intact chromosome complexes probably will not be affected during these two stages. However, they may also decay under extremely unfavourable conditions, due to a misfunction of the periplasmodium, this being the third stage.     

Senescence vs. changes in nuclear DNA, protein and dry mass contents in leaf mesophyll of two species differing in occurrence of endomitotic polyploidy

DNA and Naphtol yellow S-staining (F-NYS) protein contents were measured cytophotometrically using the Feulgen method in the nuclei of the mesophyll from the basal and apical zone of young and old leaves in two perennial monocotyledonous species: Rhoeo discolor and Clivia miniata, differing in presence or absence of DNA endoreplication. Dry mass content was determined interferometrically using an uniform field with large image shearing method. It has been shown that nuclei with 2C DNA and below 2C DNA content dominate in old leaves. The decrease in dry mass content of nuclei correlated with the decrease in NYS protein content. Parallelly a significant increase in NYS protein and DNA contents observed in chromocenters Rhoeo discolor was proportional to the increase in their dry mass. The decrease in nuclear DNA content in mesophyll of old leaves in endoreplicating species was the same as in non-edoreplicating one, however the senescence was more intensive in endoreplicating species.     

Rumex acetosa Y chromosomes: constitutive or facultative heterochromatin?

Condensed Y chromosomes in Rumex acetosa L. root-tip nuclei were studied using 5-azaC treatment and immunohistochemical detection of methylated histones. Although Y chromosomes were decondensed within root meristem in vivo, they became condensed and heteropycnotic in roots cultured in vitro. 5-azacytidine (5-azaC) treatment of cultured roots caused transitional dispersion of their Y chromosome bodies, but 7 days after removal of the drug from the culture medium, Y heterochromatin recondensed and again became visible. The response of Rumex sex chromatin to 5-azaC was compared with that of condensed segments of pericentromeric heterochromatin in Rhoeo spathacea (Sw.) Steam roots. It was shown that Rhoeo chromocentres, composed of AT-rich constitutive heterochromatin, did not undergo decondensation after 5-azaC treatment. The Y-bodies observed within male nuclei of R. acetosa were globally enriched with H3 histone, demethylated at lysine 4 and methylated at lysine 9. This is the first report of histone tail-modification in condensed sex chromatin in plants. Our results suggest that the interphase condensation of Y chromosomes in Rumex is facultative rather than constitutive. Furthermore, the observed response of Y-bodies to 5-azaC may result indirectly from demethylation and the subsequent altered expression of unknown genes controlling tissue-specific Y-inactivation as opposed to the global demethylation of Y-chromosome DNA.     

Influence of decenylsuccinic Acid on water permeability of plant cells

Decenylsuccinic acid altered permeability to water of epidermal cells of bulb scales of Allium cepa and of the leaf midrib of Rhoeo discolor. Water permeability, as determined by deplasmolysis time measurements, was related to the dose of undissociated decenylsuccinic acid (mm undissociated decenylsuccinic acid × minute). No relationship was found between permeability and total dose of decenylsuccinic acid, or dose of dissociated decenylsuccinic acid, suggesting that the undissociated molecule was the active factor in permeability changes and injury.     

Variation at Two Loci affecting Homoeologous Meiotic Chromosome Pairing in <Emphasis Type="Italic">Triticum aestivum</Emphasis> × <Emphasis Type="Italic">Aegilops mutica</Emphasis> Hybrids

HOMOEOLOGOUS chromosomes of the three genomes of bread wheat (Triticum aestivum 2n=6x=42) are normally prevented from pairing at meiosis by the activity of an allele at the Ph locus on chromosome 5B^L (refs. 1–4). This activity is responsible for the regular bivalent-forming meiotic behaviour and for the stable disomic inheritance of T. aestivum. If allelic variation occurs at the PA locus in nature it is extremely rare, although mutation has been induced and mutant alleles isolated^3,4.     

Does plasmolysis increase the drought tolerance of plant cells?

Summary Iljin's experiments withRhoeo discolor, cabbage and red beet which seemed to demonstrate increased drought resistance of plasmolysed tissue have been repeated, but his results could not be confirmed. The tissue plasmolysed in sucrose solutions, died either during stepwise plasmolysis and deplasmolysis or else later on, during exposure to unsaturated air within one or two days, even at the highest humidities. Iljin's error was apparently produced by a wrong interpretation of his tests of viability: the plasma of his dead cells did not disintegrate and retained anthocyanin by tonoplast plasmolysis.Plasmolysis proved harmful to all three objects investigated.In view of these results and earlier criticism of Iljin's analogous experiments on frost resistance by others, all experimental evidence produced in support of Iljin's mechanical theory of drought resistance should be reexamined.     

Uncoupling of sexual reproduction from homologous recombination in homozygous Oenothera species

Salient features of the first meiotic division are independent segregation of chromosomes and homologous recombination (HR). In non-sexually reproducing, homozygous species studied to date HR is absent. In this study, we constructed the first linkage maps of homozygous, bivalent-forming Oenothera species and provide evidence that HR was exclusively confined to the chromosome ends of all linkage groups in our population. Co-segregation of complementary DNA-based markers with the major group of AFLP markers indicates that HR has only a minor role in generating genetic diversity of this taxon despite its efficient adaptation capability. Uneven chromosome condensation during meiosis in Oenothera may account for restriction of HR. The use of plants with ancient chromosomal arm arrangement demonstrates that limitation of HR occurred before and independent from species hybridizations and reciprocal translocations of chromosome arms-a phenomenon, which is widespread in the genus. We propose that consecutive loss of HR favored the evolution of reciprocal translocations, beneficial superlinkage groups and ultimately permanent translocation heterozygosity.     

Identification of complexes in a spontaneous triploid Rhoeo spathacea

A ring-of-12 chromosomes at meiosis is characteristic of diploid Rhoeo. Each arm has been assigned a letter in accordance with the segmental interchange theory. Adjacent distribution at anaphase I results in nonviable spores while alternate distribution results in only two types of spores, both viable. Each of these two types has six chromosomes which are collectively either an or a complex. The chromosome complement of a diploid contains one of each. A theoretical diakinesis configuration in the spontaneous triploid Rhoeo is a ring with six bivalents alternating with six univalents. Among the twelve connecting positions, Position D is diagnostic in the triploid. If the complex is in duplicate, two short arms and a long arm are connected at Position D, and Univalent cD is attached to two bivalents by their short arms. In contrast, if the complex is in duplicate, two long arms and a short arm are connected at Position D, and Univalent Dd is attached to two bivalents by their long arms. Squashed preparations of PMC stained with acetocarmine were used. Among a larger number of triploid metaphase I cells studied, 53 had identifiable chromosomes. In four of the 53, all 18 chromosomes were identified. Chiasma failures in these four PMC were distributed at random among the 12 positions, and at random relative to arm length. The unique features predicted in the presence of an extra complex were observed. Root tip karyotypes had only four (rather than five) large metacentrics. It is concluded that the chromosome complement of the triploid consists of two complexes and one complex. Implications for the balanced lethal mechanism are discussed.This paper represents part of a dissertation submitted in partial fulfillment of the requirements for a Ph. D. degree in genetics at The Ohio State University, Columbus, Ohio, USA.     

Cultivation of Excised Anthers in vitro--Effect of Nucleic Acids

Anthers of Allium cepa and Rhoeo discolor, excised at leptotene-zygoteneand diplotene-diakinesis, have been cultured in modified White'smedium supplemented with different concentrations of ribonucleicacid and desoxyribonucleic acid. The best development was obtainedwith ribonucleric acid in which there was a 100 per cent. survivalof the microspore mother cells up to the formation of 1-celledmicrospores. In R. discolor, the time taken by the microsporemother cells to form tetrads in vitri was half of that requiredin nature. All attempts to induce division of the microsporenucleus in vitro failed.