Stimulation of onion root elongation by ascorbate and ascorbate free radical inAllium cepa L.

Summary We report that ascorbate free radical stimulates onion root growth at 15 °C and 25 °C. The fully reduced form, ascorbate, also stimulates root elongation if culture conditions allow its oxidation. When ascorbate oxidation was inhibited, no stimulation of root growth was found. The effect of the fully oxidized form, dehydroascorbate, was inhibitory. We show also that ascorbate free radical generated by ascorbate oxidation, is reduced back probably by a transplasmalemma reductase. These results are discussed on the basis of an activation of a transplasma membrane redox system likely involved in processes related to cell growth.Abbreviations AFR ascorbate free radical - ASC ascorbate - DHA dehydroascorbate     

Reversion of the antimitotic effect of N-formylbiuret by ureidosuccinic acid and uracil inAllium cepa L.

Byl sledován vliv N-formylbiuretu, degrada?ního produktu 5-azauracilu, na pr?běh mitóz v ko?enových meristémechAllium cepa. V p?ítomnosti 4. 10^-3 M koncentrace N-formylbiuretu v ?ivném roztoku dochází v ko?enových meristémech k 20–30% sní?ení mitotického indexu. Toto sní?ení mitotického indexu lze odstranit p?idáním kyseliny ureidojantarovéa uracilu do média. N-Formylbiuret, který v d?sledku interakce s dihydroorotázou inhibuje biosyntézu pyrimidinových prekursor? nukleových kyselin, nejeví tak p?i sledování cytologických ú?ink? irreversibilní toxicitu. P?i sní?ení mitotického indexu N-formylbiuretem nebylyv anafázi a v telofázi pozorovány ?ádné chromozomální aberace. Strukturálně p?íbuzné halogenacetylderiváty mo?oviny se za stejných podmínek v ?ádově ni??ích koncentracích vyzna?ují zna?nou cytotoxicitou spojenou s aglutinací chromozóm?. Zvrat antimitotického ú?inku těchto látek pyrimidinovými prekursory nukleových kyselin nebyl prokázán.     

Fine structure of synaptonemal-like complexes inAllium cepa microspores

Summary Synaptonemal-like complexes appear inAllium cepa microspores as tubular structures. They are formed of 60 nm large granules which show a finely fibrillar texture. The multiple exposure rotation technique byMarkham et al. suggests that these tubular structures are composed of twelve subunits.     

Nucleolar Organizer ultrastructure in Allium cepa

The Nuoleolar Organizer Region (NOR) was studied in Allium cepa. The cells analyzed were: meristematic root tip cells, cells from the layers of the anther (exothecium, intermediate layer cells, endothecium and tapetum) and meiocytes from preleptotene to diptotene. Conventional electron microscope techniques and serial section were used in the study. Ultracytochemical tests, using preferential techniques for nucleic acids were also applied. The results show that: a) Although the NOR may have a very variable form in the different cells analyzed, in each case its fine structure is similar, being constituted of chromatin differentiated in high and low density zones. b) In each case, its cytochemical characteristics are similar; highly positively to EDTA (preferential stain for RNP) being observed in the low density zones. As scattered chromatin these would constitute the active regions of the NOR. c) In certain cases a clear relationship is seen between NOR volume and the size of the nucleolus. d) The chromatin associated to the NOR always appears as very dense zones. e) The NOR-Nucleolus relationships are established exclusively with the latter's fibrillar pars. f) The nucleolar segregation phenomenon takes place sometimes as a result of a NOR contraction and in other cases depends on the position of the NOR in relation to the nucleolus. g) In preleptotene, the NOR shows its characteristic morphology and is positive to EDTA, while from zygotene to diplotene the structure of the NOR presents no differences from the rest of the chromatin and only the lateral elements of the synaptonemal complex or its remnants are positive to EDTA. h) The synaptonemal complex runs through the NOR with no differentiation and on occasions its lateral elements show associations with the nucleolus.     

Nucleolar organizing chromosomes ofRicinus

Summary Pachytene chromosome morphology was compared in nine races ofRicinus communis L. (2n = 20), using pollen mother cells (PMCs) and light microscopy. Of the ten bivalents, only the two possessing nucleolar organizing regions (NORs), chromosomes 2 and 7, exhibit structural variations among the races. The NORs are located in the short arms of these two chromosomes. Most of the observed structural variations affect these short arms, which are similar morphologically and consist largely of heterochromatic segments. The PMCs contain a single nucleolus and this is associated with the NOR of each of the two chromosomes at a particular frequency in each race. In eight races, a nucleolar constriction (NC) is present in either chromosome 2 or chromosome 7. In these races, the nucleolus is associated with the chromosome possessing an NC at a frequency of 100% and with the chromosome lacking an NC at a frequency ranging between 5.6 and 100%, depending upon the race. No microscopically visible NC is present in the ninth race. In this race, the nucleolus is associated with both chromosomes 2 and 7 at a frequency of 100%. The association of the nucleolus with a chromosome possessing an NC is at the NC and with a chromosome lacking an NC is at the terminal heterochromatic segment of the short arm. Several interpretations are offered to account for the variations in frequency of association between the nucleolus and each of the nucleolar organizing chromosomes. It is suggested that the two non-linked NORs have evolved through some intragenomic changes rather than polyploidy, that this species is highly intolerant to structural variations other than those occurring in or near the NORs, and that structural variations in the nucleolar organizing chromosomes are not associated with racial variations in plant phenotype.Paper of the Journal Series, New Jersey Agricultural Experiment Station     

Nucleolar organizer regions (NORs) in Chinese hamster chromosomes as visualized by Coomasie brilliant blue

Nucleolar oragnizer regions (NORs) of Chinese hamster chromosomes have been demonstrated by using a Coomasie brilliant blue dye (CBB) method. The staining procedure involved is simple and the results are reproducible. The staining process is easily controllable because over-staining of the chromosomes seldom occurs. The CBB solution is stable (pH 3) and can be used for many days at room temperature. Contrary to the silver technique, the stained material in the NORs is resistant to acid extraction. Since it is sensitive to trypsin treatment, it is suggested that the CBB stained material is protein in nature.     

Sequence heterogeneity of the ribosomal DNA inAllium cepa (Liliaceae)

Summary Southern blot hybridizations were performed to investigate the ribosomal DNA structure inAllium cepa (Liliaceae). The analysis of single and double digestions with Eco RI, Hind III, and Bam HI endonuclease restriction enzymes showed the presence of at least two types of repetitive units. The gene types were present in each single individual ofAllium cepa analyzed, notwithstanding the variation of the ribosomal gene number from 5,500 to 11,900 and the NOR number from 2 to 4. The first and the second gene type are 12.7 kb long and the first type is much more represented inAllium cepa genome. The differences between the two gene types consist in the position of the Hind III restriction sites in the external spacer.Work supported by CNR contract no. 74/0267.     

Chromosome organization revealed upon the decondensation of telophase chromosomes inAllium

Chromosomes of root tip cells ofAllium cepa andAllium sativum were studied in early, middle and late telophase to examine the organization of mitotic chromosomes, taking advantage of the naturally occurring chromosome dispersion during the process of decondensation in telophase. Longitudinal and transverse sections of telophase chromosomes viewed under the transmission electron microscope showed that mitotic chromosomes inAllium were composed of helically coiled 400–550 nm chromatin fibres. In some regions of the longitudinal sections, these chromatin fibres were seen to be orientated parallel to one another but formed roughly a right angle to the long axis of the chromosome. In transverse sections, the telophase chromosome appeared to have a hollow centre encircled by the 400–550 nm chromatin fibre which in turn was a hollow tube structure formed by the coiling of a thinner fibre of 170–200 nm. In addition, cross views of chromatin fibres of 170–200 nm and 50–70 nm were also identified in telophase chromosome preparations. These two organizational levels of chromatin fibres also showed a hollow centre. The process of decondensation of telophase chromosomes is described, and some morphological characteristics associated with the activities of chromosome decondensation are analysed. Based on the observations made onAllium chromosomes in this study, various models of chromosome organization are discussed.     

Nucleolar organizer region variants as a risk factor for Down syndrome.

An unusual nucleolar organizer region (NOR) heteromorphism was noted among 13 of 41 parents in whom nondisjunction leading to trisomy 21 was known to have occurred. In contrast, only one of these double NOR (dNOR) variants was found among the 41 normal spouses and none were seen among 50 control individuals. In two dNOR(+) families, a second child with trisomy 21 was conceived. In both families, the extra chromosome in each child was contributed by the parent who carried the dNOR variant and resulted from a recurrent meiosis I error. Our data suggest that the dNOR heteromorphism may play a role in meiotic nondisjunction and could be associated with as much as a 20-fold increased risk for having offspring with trisomy 21.     

Nucleolar organizing regions of human chromosomes

Summary Silver-Stained cells from 49 parents with a history of several abortions were compared with cells from 35 parents with normal liveborn children. The modal and mean number of silver-stained NORs (Ag-NORs) observed on D- or G-group chromosomes was similar in both groups and between males and females. Ag-NORs were randomly distributed on all five acrocentric pairs. The distribution and size of Ag-NORs within an individual was not random and was fairly consistent from cell to cell.The mean number of associations per cell was similar in both males and females of the abortion group and was less than the number of associations in controls. The probability of D- or G-group chromosomes being associated was near the expected probability of 0.6 for D-association and 0.4 for G-association. The frequency of association of any chromosome combination did not differ statistically from the expected values, though the number of associations, 15/22, was higher than expected.     

AFLP linkage group assignment to the chromosomes of Allium cepa L. via monosomic addition lines

Two complete sets of Allium fistulosum L.– A. cepa monosomic addition lines (2n=2x+1=17) together with an AFLP linkage map based on a cross between A. cepa and A. roylei Stearn were used to re-evaluate the eight A. cepa linkage groups identified in the mapping study. The linkage groups could be assigned to individual, physical chromosomes. The low level of molecular homology between A. cepa and A. fistulosum enabled the identification of 186 amplified fragment length polymorphisms (AFLP™ markers) present in A. cepa and not in A. fistulosum with ten different primer combinations. With the monosomic addition lines the distribution of the markers over the eight chromosomes of A. cepa could be determined. Of these 186 AFLP markers 51 were absent in A. roylei and consequently used as markers in the mapping study (A. cepa ×A. roylei cross). Therefore, these 51 AFLP markers could be used to assign the eight A. cepa linkage groups identified in the mapping study to physical chromosomes. Seven isozyme and three CAPS markers were also included. Two of the linkage groups had to be split because they included two sets of markers corresponding to different chromosomes. A total of 20 (approx. 10%) of the A. cepa-specific AFLP markers were amplified in more than one type of the monosomic addition lines, suggesting unlinked duplications. The co-dominant isozyme and CAPS markers were used to identify the correspondence of linkage groupsoriginating from A. cepa or from A. roylei. Received: 16 April 1999 / Accepted: 13 August 1999     

Recombinant chromosomes of advanced backcross plants between Allium cepa L. and A. fistulosum L. revealed by in situ hybridization

Cytological analysis of (Allium cepa L.×Allium fistulosum L.)×A. cepa L. F₁BC₃ plants revealed most plants were diploid with 16 chromosomes. Karyotypes of these plants showed recombinant chromosomes. Fluorescence and genomic in situ hybridization patterns of interspecific F₁ hybrid and F₁BC₃ plants revealed A. fistulosum chromosomes or chromosomal segments. A highly repetitive 376-bp DNA sequence and genomic DNA of A. fistulosum revealed similar telomeric hybridization sites when hybridized onto A. fistulosum chromosomes. Cytogenetic evidence showed that A. fistulosum DNA has recombined into the A. cepa genome. Received: 20 October 1999 / Accepted: 11 November 1999     

Relationship between secondary constrictions and nucleolus organizing regions inAllium sativum chromosomes

Summary Allium sativum L. (2 n=16) had three types of clones with regard to the number of chromosomes carrying well-defined secondary constrictions: the first type had two secondary constricted chromosomes (type I), the second had three (type II) and the third had four (type III). Silver staining was applied to these three types of cells to determine the number of nucleolus organizing regions (NORs) per cell and to study the relationship between the morphological appearance of the secondary constrictions and the ability of the chromosomes to form nucleoli. Ag-positive regions appeared on two chromosomes in type I, on three in type II and on four in type III. The comparison of Giemsa and Feulgen stained chromosomes with the silver stained ones clearly indicated that the positive reaction with silver occurred exclusively on the secondary constricted regions that responded negatively to both Giemsa and Feulgen staining, indicating that the size of the achromatic secondary constrictions directly reflects the volume of the Ag-positive materials. However, all three types of clones had a maximum of four nucleoli at interphase. Of the four nucleoli, either two or one was extremely small (less than 1 m in diameter) in types I and II respectively. The size variations of the other nucleoli seemed to be positively correlated with those of the Ag-positive regions. This and the observation that the maximum number of nucleoli per cell did not coincide with the number of Ag-positive regions on the metaphase chromosome complement suggest strongly that the NORs responsible for the minute nucleoli cannot be detected on the metaphase chromosomes. The present observations indicate that not all NORs are indicated by the morphological appearance of secondary constrictions.     

Nucleolar structure during the meiotic prophase in Allium cepa anthers

Summary Naturally segregated nucleoli have been observed during the prophase in meiocytes of Allium cepa anthers. Under the light microscope the nucleolus is seen to consist of two clearly differentiated regions: a central core, which is strongly argyrophilic (ochre or dark brown) and slightly basophilic, surrounded by a basophilic peripheral region, which shows a low degree of argyrophilia. Under the electron microscope the central region appears as consisting of fibrillar elements (pars fibrosa), while the peripheral region proved to consist mainly of granules about 150 Å in diameter (pars granulosa).When the pachytene nucleolus of Allium cepa is stained with basic fuchsin, a small circular area appears intensely stained, which gradually grows larger as the pachytene proceeds. This characteristic structure, eventually reaching a size of 0.5–1.5 is regularly to be observed with a central vacuole. Under the electron microscope this area appears as a circular structure of high electron density, which corresponds in shape and size with the area revealed by the light microscope.The relationship between this new structure, which we have called globulus with other nucleolar structures is discussed.This work was partially supported by a grant from the Fondo de Ayuda a la Investigación, 1968, Spain. We wish to thank most especially Dr. M. C. Risueño, M.I. Rodriguez-García and J. M. Sogo, of the Cell Structures Section, (Department of Cytology) for their efficient collaboration. We also wish to thank M. C. Partearroyo and A. Partearroyo for technical assistance. One of the authors (J.C.S.) has a Research Training Fellowship awarded by the International Agency for Research on Cancer (World Health Organization).     

The chromosomes and DNA of Allium cepa

Giemsa C-banded idiograms that allow the identification of all chromosomes have been prepared for Allium cepa, Ornithogalum virens, and Secale cereale. An analysis of A. cepa DNA has determined that: (1) It has the lowest GC content so far reported for an angiosperm (32%). (2) It appears to have no satellite DNA detectable by CsCl or Cs₂SO₄-Ag⁺ density gradient centrifugation. (3) Aside from fold back DNA and unreactable fragments, a C₀t curve indicates that most of the DNA can be adequately described as two major middle repetitive components (Fractions I and II) and a single copy component (Fraction III). And (4) most of the repeated DNA sequences are involved in a short period interspersion pattern with single copy and other repetitive sequences. In situ hybridization of tritiated cRNAs to fold back, long repeated, and Fraction I DNA from A. cepa to squash preparations of chromosomes and nuclei from A. cepa, O. virens, and S. cereale root tips indicates: (1) Sequences complementary to fold back DNA are scattered throughout the genome of A. cepa except for telomeric heterochromatin and nucleolus organizers while they are not detectable in the genomes of O. virens or S. cereale. (2) Although long repeated sequences are scattered throughout the genome of A. cepa, they are concentrated to some extent in telomeric heterochromatin and nucleolus organizers (NOs). Sequences complementary to long repeats of A. cepa occur primarily in chromosome three of O. virens while these sequences are more common in the genome of more distantly related S. cereale. (3) Fraction I DNA is scattered throughout the genome of A. cepa while it is hardly detectable in the genomes of O. virens and S. cereale. These results are discussed in regard to the evolutionary conservation and function of repeated DNA sequences.