The effect of lead on the cell cycle in the root meristem ofAllium cepa L.

Summary Allium cepa (L.) adventitious roots were treated with lead (2.5 mg of Pb²⁺ [from Pb(NO₃)₂] per dm³) for 30–72 h. The cell cycle was studied by pulse labeling with [³H]thymidine. Mitotic activity kinetics, occurrence of disturbed mitoses (c-mitoses), and level of DNA synthesis were examined. It was found that lead prolonged the cell cycle and that cells in two phases of the cycle, G₂ and S, differed in their sensitivity to lead. Cells in G₂ were more sensitive; lead lengthened their cycle by 216% and disturbed the course of cell division by causing c-mitoses. Cells in S phase were less sensitive. Their cell cycle was longer by 55%. They went through their G₂ phase without major disturbances, mitosis in these cells was normal. During treatment ofA. cepa with lead, its destructive effects on cells were exerted only during the first few hours (around 6 h) of incubation. That is when the inhibition of mitotic activity, numerous disturbances of cell division, a decline in the number of cells synthesizing DNA, and a lower level of DNA synthesis were observed. As the incubation continued, the above processes were found to return to normal. In the discussion, data are presented supporting the hypothesis that during the initial period of exposure ofA. cepa to lead, this metal enters both the root apoplast and symplast, exerting a destructive effect on cells, while later, lead penetrates only into the root apoplast, and in this way remains harmless to cells.     

Anti-mitotic and anti-genotoxic effects of Plantago lanceolata aqueous extract on Allium cepa root tip meristem cells

Plantago is the most important genus of Plantaginaceae family and is used in traditional medicine around the world for different purposes. Plantago coronopus L., Plantago major L., Plantago media L. and Plantago lanceolata L. are most commonly used species of Plantago in traditional medicine in Turkey. The main goal of this study was to investigate the eventual anti-mitotic and anti-genotoxic effects of P. lanceolata L. leaf aqueous extracts (15 g/L and 30 g/L) on Allium cepa L. root tip meristem cells which were treated with 0.7% hydrogen peroxide. For this purpose, two different experiments were performed under the same conditions. In the first experiment, Allium cepa onion bulbs were treated with 0.7% H₂O₂ for 1 h. After the H₂O₂ treatment, the onion bulbs were treated with two different concentrations (15 g/L and 30 g/L) of P. lanceolata extracts for 24 h. In the second experiment, A. cepa onion bulbs were treated with two different extract concentrations (15 g/L and 30 g/L) for 24 h and then with 0.7% H₂O₂ for 1 h. The test concentrations were determined according to doses which are recommended in alternative medicinal usage by people. As positive and negative control 0.7% H₂O₂ and tap water was used, respectively. As a result, it was determined that aqueous extracts reduced mitotic index and chromosome aberrations in treatment groups in comparison with controls. These results showed that P. lanceolata aqueous extracts have anti-mitotic and anti-genotoxic effects.     

Behavior of leucoplast nucleoids in the epidermal cell of onion (Allium cepa) bulb

Summary The behavior of nucleoids during the leucoplast division cycle in the epidermis of onion (Allium cepa) bulbs was investigated using DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) staining. The leucoplast was morphologically amoeboid and continuously changed its shape. A dumbbell-shaped leucoplast divided into two spherical daughter ones by constriction in the middle region of the body. Leucoplasts contained 4–10 mostly spherical, oval, partly rodand dumbbell-shaped nucleoids which were dispersed within the bodies. The proportion of one DNA molecule of a T4 phage particle to the small leucoplast nucleoid in the grain density of negative film was 1 to 0.91. Comparison of the present result and another groups' biochemical results suggested that a small leucoplast nucleoid contains one DNA molecule. The dumbbell-shaped leucoplast probably before division contained about twice as many nucleoids as the spherical leucoplast after division, and each half of the dumbbell contained about half the number of nucleoids. Nucleoids increased in number with growth of the leucoplast. The behavior of nucleoids during the leucoplast division cycle in onion bulbs was basically similar to that during the chloroplast division cycle in higher plants and green algae, which was previously reported (Kuroiwa et al. 1981 b).     

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).     

Plasmalemma-associated malate dehydrogenase activity in onion root cells

Summary Plasma membrane vesicles isolated from onion roots showed oxaloacetate reductase activity as well as other oxidoreductase activities. Purification and further sequencing showed that the protein responsible for the activity is a 40 kDa protein which corresponds to the cytosolic soluble malate dehydrogenase. However, the activity remained bound to the membrane after repeated freezing and thawing cycles and further washing, excluding a cytosolic contamination as the source of the activity. Furthermore, a second 28 kDa protein has been copurified together with the 40 kDa protein. The plasmalemma oxaloacetate reductase activity shows both donor and acceptor sites located towards the cytoplasmic side of the plasma membrane. This enzyme catalyzed the oxidation of NADH by oxaloacetate and the reduction of NAD⁺ by malate in the presence of an oxaloacetate-withdrawing system. We conclude that a significant amount of the cytosolic malate dehydrogenase can be specifically attached to the cytosolic face of the plasmalemma. A possible role in a putative malate shuttle associated to the plasma membrane is discussed.Abbreviations AFR ascorbate free radical - DQ duroquinone - OA oxaloacetate - DPIP dichlorophenolindophenol - MDH malate dehydrogenase - PHMB p-hydroxymercuribenzoate     

Fidelity of Hymenoptera and Diptera pollinators in onion (Allium cepa L.) pollination

Onion (Allium cepa L.) is protandrous in nature and requires cross‐pollination to avoid inbreeding. The pollination potential of native bees (Hymenoptera) and true flies (Diptera) was assessed in the perspective of finding the best pollinators for onion cross‐pollination and seed multiplication. The community of pollinators was composed of four bee species and twelve true fly species. Episyrphus balteatus, Eupeodes sp., Musca domestica and Eristalinus aeneus were the most abundant pollinators. The maximum pollinator activity was observed from 12 to 24 days after opening of the flowers. The pollination effectiveness of tested bees (Apis dorsata and Apis florea) was greater than true flies (E. balteatus, Eupeodes sp., M. domestica, E. aeneus and Callihoridae sp.) in terms of Spears values.     

Development of the preprophase band from random cytoplasmic microtubules in guard mother cells of Allium cepa L.

The organization of microtubule (MT) arrays in the guard mother cells (GMCs) of A. cepa was examined, focussing on the stage at which a longitudinal preprophase band (PPB) is established perpendicular to all other division planes in the epidermis. In the majority of young GMCs, including those seen just after asymmetric division, MTs are distributed randomly throughout the cortex and inner regions of the cytoplasm. Few MTs are associated with the nuclear surface. As the GMCs continue to develop, MTs cluster around the nucleus and a PPB appears as a wide longitudinal band. Microtubules also become prominent between the nucleus and the periclinal and transverse walls, while they decrease in number along the radial longitudinal walls. The PPB progressively narrows by early prophase, and a transversely oriented spindle gradually ensheaths the nucleus. These observations indicate that the initial, broad PPB is organized by a rearrangement of the random cytoplasmic array of MTs. Additional reorganization is responsible for MTs linking the nucleus and the cortex in the future plane of the cell plate, and for narrowing of the PPB.Abbreviations GMC guard mother cell - MT microtubule - PPB preprophase band     

Loosening of a preprophase band of microtubules in onion (Allium cepa L.) root tip cells by kinase inhibitors

Effects of kinase inhibitors on the preprophase band of microtubules in onion (Allium cepa L.) root tip cells were examined. Bundled microtubules in preprophase bands were dispersed on the cell cortex when onion seedlings were incubated with 2.5-5.0 mM 6-dimethylaminopurine. Fifteen min was enough for the bundled microtubules to disappear. Although many preprophase bands remained when the seedlings were incubated with 60 microM staurosporin, these preprophase band microtubules were loosened and the width of the band became broad. These results sugget that some kinases are involved in the microtubule bundling in the preprophase band development.     

Abundance of microtubules in preprophase bands of some Triticum species

Root tip procambial cells of Triticum speltoides, T. tauschii, T. turgidum and T. aestivum have been investigated ultrastructurally for the detection of preprophase microtubule bands (PMBs) and to estimate the number of microtubules comprising the bands. The species selected are phylogenetically related but differ in the ploidy level. It was found that all species develop well-defined PMBs prior to mitosis. Estimations of microtubule abundance in the PMBs was carried out in midpreprophase cells, a stage judged by a feature of the nucleus in which electron-transparent canals are formed around the initial condensations of the chromatin material and the nucleoli. Triticum speltoides bears the smaller average number of microtubules per PMB and T. aestivum the greater. The results indicate that the increase follows the upgrade of the number of chromosome sets. It is suggested that the average number of microtubules of PMBs is related to the ploidy level.Abbreviation PMB preprophase microtubule band     

The role of Ca2+ in elicitation of phytoalexin synthesis in cell culture of onion (Allium cepa L.)

Summary Treatment of Allium cepa L. cellsuspension cultures with a biotic elicitor derived from the fungus Botrytis cinerea, resulted in phytoalexin synthesis. Two phytoalexins, 5-octylcyclopenta-1,3-dione and 5-hexyl-cyclopenta-1,3-dione, were accumulated in cultured onion cells. Removal of extracellular Ca²⁺ by the calcium chelator ethylene glycol bis(b-aminoethyl ether) N,N-tetraacetic acid abolished the elicitor-mediated phytoalexin synthesis. The calcium channel blockers, verapamil and 8-N,N-(dimethylamino)octyl-3,4,5-trimethoxybenzoate caused similar effects, whereas the addition of the Ca²⁺ ionophore A23187 enhanced the accumulation of phytoalexins in the absence of the elicitor. Increase in the cytoplasmic Ca²⁺ concentration in elicitor-treated onion cells was observed as monitored by the fluorescent calcium indicator indo-1. These observations suggest that Ca²⁺ acts as a second messenger in the regulation of phytoalexin synthesis in cultured onion cells.Abbreviations A23187 4-bromo-calcium ionophore - cAMP adenosine 3,5-cyclic monophosphate - [Ca²⁺]_cyt cytoplasmic Ca²⁺ concentration - EGTA ethylene glycol bis(b-aminoethyl ether) N,N-tetraacetic acid - EtOH ethanol - Et₂O diethyl ether - fr.wt fresh weight - HR hypersensitive response - PIPES piperazine N,N-bis-(2-ethanesulfonic acid) - TMB-8 [8-N,N-(dimethylamino)] octyl-3,4,5-trimethoxy-benzoate - Tsl tsibulin     

Structural studies of microsporogenesis in fertile and male-sterile onions (Allium cepa L.) containing the cms-S cytoplasm

Summary The structure of anther tissues has been studied during microsporogenesis in male-sterile and -fertile onions. Three types of abnormal tapetal behaviour have been observed within the single line II/3ms containing the cms-S cytoplasm: type 1, the premature breakdown of the tapetum at the tetrad stage, type 2, the hypertrophy of the tapetum after the diad stage followed by its premature autolysis and, type 3, in which the tapetum remains in good condition but for an abnormally long period of time. Tapetal autolysis proceeds in the same manner in both male-steriles and -fertiles with only the stage at which it occurs differing between the types of plants. Mitochondria were prominent in the tapetal tissue of all onion types throughout all stages of microsporogenesis and were still visible during the last stages of tapetal autolysis. In a detailed study of type 2 behaviour, no differences in mitochondrial volumes were found until the tapetum hypertrophied.     

Plastid apportionment and preprophase microtubule bands in monoplastidic root meristem cells ofIsoetes andSelaginella

Summary Ultrastructural observations on monoplastidic root tip cells ofIsoetes andSelaginella demonstrate two important phenomena associated with preprophasic preparation for mitotic cell division, 1. the preprophase band and 2. precise orientation of the dividing plastid relative to the preprophase band. Both of these phenomena accurately predict the future plane of cell division. The plastid divides in a plane parallel to the spindle and each cell inherits a single plastid which caps the telophase nucleus. When succesive transverse divisions occur, the plastid migrates prior to prophase from a position near an old transverse wall to a lateral position in the cell. The plastid is oriented with its median constriction precisely intersected by the plane of the preprophase band. When a longitudinal division follows a transverse division, the plastid remains in its position adjacent to an old transverse wall where it is bisected by the plane of the longitudinally oriented preprophase band microtubules.     

Preprophase bands of microtubules and the cell cycle: Kinetics and experimental uncoupling of their formation from the nuclear cycle in onion root-tip cells

We have studied the timing of preprophase band (PPB) development in the division cycle of onion (Allium cepa L.) root-tip cells by combinations of immunofluorescence microscopy of microtubules, microspectrophotometry of nuclear DNA, and autoradiography of [³H]thymidine incorporation during pulse-chase experiments. In normally grown onion root tips, every cell with a PPB had the G2 level of nuclear DNA. Some were in interphase, prior to chromatin condensation, and some had varying degrees of chromatin condensation, up to the stage of prophase at which the PPB-prophase spindle transition occurs. In addition, autoradiography showed that PPBs can be formed in cells which have just finished their S phase, and microspectrophotometry enabled us to detect a population of cells in G2 which had no PPBs, these presumably including cells which had left the division cycle. The effects of inhibitors of DNA synthesis showed that the formation of PPBs is not fully coupled to events of the nuclear cycle. Although the mitotic index decreased 6-10-fold to less than 0.5% when roots were kept in 20 g·ml^-1 aphidicolin for more than 8 h, the percentage of cells containing PPBs did not decrease in proportion: the number of cells in interphase with PPBs increased while the number in prophase decreased. Almost the same phenomena were observed in the presence of 100 g·ml^-1 5-aminouracil and 40 g·ml^-1 hydroxyurea. In controls, all cells with PPBs were in G2 or prophase, but in the presence of aphidicolin, 5-aminouracil or hydroxyurea, some of the interphase cells with PPBs were in the S phase or even in the G1 phase. We conclude that PPB formation normally occurs in G2 (in at least some cases very early in G2) and that this timing can be experimentally uncoupled from the timing of DNA duplication in the cell-division cycle. The result accords with other evidence indicating that the cytoplasmic events of cytokinesis are controlled in parallel to the nuclear cycle, rather than in an obligatorily coupled sequence.Abbreviations APC aphidicolin - 5-AU 5-aminouracil - DAPI 4, 6-diamidino-2phenylindole - HU hydroxyurea - MI mitotic index - MT microtubule - PMSF phenylmethyl-sulfonyl fluoride - PPB preprophase band - %PPB percentage of cells with PPBs     

Effects of phosphorus on the morphology of VA mycorrhizal root system of leek (Allium porrum L.)

Leek plants were raised on sterilized soil/sand medium amended with Ca(H₂PO₄)₂. H₂O ranging up to 750 mg Pkg⁻¹. Addition of P increased total root length by increasing the initiation of laterals, which extended at a constant rate. This change in root growth was paralleled by the P inflow into the plant, but not by the P concentration of the plant, suggesting that it is the rate of P uptake which affects root branching.     

Allozyme variation within and among populations of onion (Allium cepa L.) from West Africa

Local germplasm of onion (Allium cepa L.) in West Africa is threatened by extinction. Sixteen populations of onion collected in five countries in West Africa were investigated for isozyme polymorphism using four polymorphic enzyme systems (ADH, MDH, 6-PGDH and PGI) among nine enzyme systems assayed. This is the first report on the genetic diversity of local landraces of onion. The inheritance of two dimeric enzyme systems PGI and MDH was demonstrated using F₂ progeny arrays. The PGI system revealed a single locus with three alleles, and the MDH system revealed three loci with four alleles. Four polymorphic systems revealed nine alleles (adh-a1 and a2, mdh-c1 and c2, 6-pgdh-a1 and a2, and pgi-a1, a2 and a3) in the 16 local populations observed. The mean number of alleles per polymorphic locus was 2.25, and 67% of the alleles were present in all populations. Allele 6-pgdh-a2 was present in only two landraces (from Niger and Nigeria); it is considered to be a rare allele (frequency approximately 2%). Among the 16 populations, within-population diversity was greater (90%) than between-population diversity (10%). Genetic distance analyses showed an aggregate of all populations except for two, which originated from Nigeria, an English-speaking country. Received: 24 August 1995 / Accepted: 26 February 2001     

Observations of the pattern of secondary wall development in the hypodermis of onion (Allium cepa) roots

Summary This brief communication reports the appearance, under certain circumstances, of root hypodermal cells with transfer cell labyrinths. These cells lie at regular intervals around the hypodermis at the bases of onion bulb roots. They are narrower (smaller tangential dimension) than unmodified cells but have the same radial dimension. These narrow cells contain small vacuoles, their main volume being composed of a cytoplasm rich in organelles, especially mitochondria. When treated with a low concentration of lanthanum nitrate solution, the tracer accumulates in the outer tangential wall and in small vacuoles and vesicles.     

Molecular and biochemical characterisation of a serine acetyltransferase of onion, Allium cepa (L.)

We have previously cloned a cDNA, designated SAT1, corresponding to a gene coding for a serine acetyltransferase (SAT) from onion (Allium cepa L.). The SAT1 locus was mapped to chromosome 7 of onion using a single-stranded conformation polymorphism (SSCP) in the 3' UTR of the gene. Northern analysis has demonstrated that expression of the SAT1 gene is induced in leaf tissue in response to low S-supply. Phylogenetic analysis has placed SAT1 in a strongly supported group (100% bootstrap) that comprises sequences that have been characterised biochemically, including Allium tuberosum, Spinacea oleracea, Glycine max, Citrullus vulgaris, and SAT5 (AT5g56760) of Arabidopsis thaliana. This group can be divided further with the SAT1 of A. cepa sequence grouping strongly with the A. tuberosum sequence. Translation of SAT1 from onion generates a protein of 289 amino acids with a calculated molecular mass of 30,573 Da and pI of 6.52. The conserved G277 and H282 residues that have been identified as critical for L-cysteine inhibition are observed at G272 and H277. SAT1 has been cloned into the pGEX plasmid, expressed in E. coli and SAT activity of the recombinant enzyme has been measured as acetyl-CoA hydrolysis detected at 232 nm. A Km of 0.72 mM was determined for l-serine as substrate, a Km of 92 microM was calculated with acetyl-CoA as substrate, and an inhibition curve for L-cysteine generated an IC50 value of 3.1 microM. Antibodies raised against the recombinant SAT1 protein recognised a protein of ca. 33 kDa in whole leaf onion extracts. These properties of the SAT1 enzyme from onion are compared with other SAT enzymes characterised from closely related species.     

Plastid transformation in greening scales of the onion bulb (Allium cepa,Alliaceae)

The greening of the upper part of the outerAllium cepa L. bulb scales, in particular along the vascular regions, is limited to the hypodermal cells in which typical leucoplasts are transformed to normal and functional chloroplasts. This process is light dependent and cannot afterwards be reversed or modified by darkness. The changes in fine structure are described and briefly discussed.Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday and 55 years after the publication of his Grundriß der Cytologie.     

Plasmodesmata in onion (Alliurn cepa L.) roots: a study enabled by improved fixation and embedding techniques

Summary Onion (Allium cepa L. cv. Ebeneezer) roots from vermiculite culture were examined with transmission electron microscopy to detect the plasmodesmata in all tissues. In young root regions, plasmodesmata linked all living cells together in all directions. In old zones, the plasmodesmatal connections of the endodermis to its neighbor tissues were not interrupted by later suberin lamella and cellulosic wall deposition. Moreover, plasmodesmata in the fully mature endodermis usually exhibited a large central cavity. In the exodermis, however, upon deposition of suberin lamellae in long cells, all plasmodesmata that initially linked them to their adjacent cells were severed. Afterwards, the long cells lost the capability of forming wound pit callose and their protoplasts began to degenerate. The mature exodermal layer was symplastically bridged to its neighbors only by the short (passage) cells that lacked suberin lamellae. Compared to the long cells, the short cells not only had thicker cytoplasm surrounding their central vacuoles but also a higher density of mitochondria and rough endoplasmic reticulum, consistent with an active involvement in the transport processes of the root. The above results were obtained by an improved, extended transmission electron microscopy procedure devised to analyze plasmodesmata in cells with suberin lamellae. By prefixing root tissues in glutaraldehyde and acrolein, all cells were well preserved. Postfixation was carried out in osmium tetroxide at a low concentration (0.5%). Following dehydration in acetone and transfer to propylene oxide, infiltration with Spurr's resin was accomplished by incubating samples in the accelerator-free mixture for 4 days, then infiltrating samples in the accelerator-amended mixture for additional 4 days.Abbreviations IE immature exodermis - ME mature exodermis - TBO toluidine blue O - TEM transmission electron microscopy     

Cytotoxic and genotoxic effects of Br-containing oxaphosphole on Allium cepa L. root tip cells and mouse bone marrow cells

The continuous production and release of chemicals into the environment has led to the need to assess their genotoxicity. Numerous organophosphorus compounds with different structures have been synthesized in recent years, and several oxaphosphole derivatives are known to possess biological activity. Such chemical compounds may influence proliferating cells and cause disturbances of the genetic material. In this study, we examined the cytotoxicity and genotoxicity of 4-bromo-N,N-diethyl-5,5-dimethyl-2,5-dihydro-1,2-oxaphosphol-2-amine 2-oxide (Br-oxph). In A. cepa cells, Br-oxph (10(-9) M, 10 (-6) M and 10 (-3) M) reduced the mitotic index 48 h after treatment with the two highest concentrations, with no significant effect at earlier intervals. Mitotic cells showed abnormalities 24 h and 48 h after treatment with the two lowest concentrations but there were no consistent changes in interphase cells. Bone marrow cells from mice treated with Br-oxph (2.82 x 10 (-3) μg/kg) also showed a reduced mitotic index after 48 h and a greater percentage of cells with aberrations (principally chromatid and isochromatid breaks). These findings indicate the cytotoxicity and genotoxicity of Br-oxph in the two systems studied.