On the properties of fluorescing compounds in guard and epidermal cells of Allium cepa L.

Onion guard cells, in contrast to those of Vicia and Pisum, do not require an alkaline treatment in order to fluoresce. Fluorescing compounds of Allium cepa L. were characterized using in-vivo microspectrophotometry; furthermore, invitro chemical analysis for epidermal tissue, intact guard and epidermal cells, and isolated guard-cell protoplasts was performed. The emission intensity (_max 520 nm) decreased when intact onion guard cells were excited with 436 nm light, but increased (_max 470 nm) when excited at 365 nm. This photodecomposition at 436 nm is typical of flavins or flavoproteins whereas an increase in fluorescence intensity with excitation at 365 nm may be explained by the presence of other substances. The presence of flavins could not be unambiguously confirmed from these results. Indeed, the absorption spectra of the vacuolar area of guard cells did not show the peak at 445 nm which is characteristic for flavins. Furthermore, there was no decrease of absorption at the excitation wavelengths of 440 and 330 nm. Since spectral data indicate the presence at high amounts of flavonoids in guard and epidermal cells, this may reduce the sensitivity for the detection of flavins in guard cells. Using thin-layer chromatography and high-performance liquid chromatography together with hydrolytic procedures, flavonol glycosides with kaempferol and quercetin as aglycones substituted with sulphate and glucuronate were identified. Further studies on guard-cell metabolism should consider the presence of flavonoids in stomata of onion and other plants.Abbreviations GCP guard-cell protoplast - HPLC high-performance liquid chromatography - TLC thin-layer chromatography     

Potassium Chloride as Stomatal Osmoticum in Allium cepa L., a Species Devoid of Starch in Guard Cells

K⁺ and Cl⁻ contents of guard cells and of ordinary epidermal cells were determined in epidermal samples of Allium cepa L. by electron probe microanalysis; malate contents of the same samples were determined by enzymic oxidation. KCl was, in general, the major osmoticum in guard cells, irrespective of whether stomata had opened on leaves or in epidermal strips floating on solutions. The solute requirement varied between 50 and 110 femtomoles KCl per micrometer increase in aperture per pair of guard cells. Stomata did not open on solutions of K iminodiacetate, presumably because its anion could not be taken up. Stomata opened if KCl or KBr was provided. Taken together, the results indicate that the absence of starch from guard cells deprived them of the ability to produce malate in amounts of osmotic consequence and that the presence of absorbable Cl⁻ (or Br⁻) was necessary for stomatal opening.     

Alien genes introgression and development of alien monosomic addition lines from a threatened species, Allium roylei Stearn, to Allium cepa L.

To produce alien monosomic addition lines (AMALs) of Allium cepa (genomes CC, 2n = 2x = 16) carrying extrachromosomes from Allium roylei (RR, 2n = 2x = 16), reciprocal backcrossing of allotriploids (2n = 24, CCR) with diploids (2n = 16, CC) and selfing of a single allotriploid were carried out. The chromosome numbers in the BC₂F₁ and BC₁F₂ progenies ranged from 16 to 32. Forty-eight plants were recorded to possess 2n = 17 among a total of 169 plants in observation. Through the analyses of isozymes, expressed sequence tag (EST) markers, and karyotypes, all eight possible types of A. cepa–A. roylei monosomic addition lines (CC+1R–CC+8R) could be identified. Seven types of representative AMALs (without CC+2R) were used for the GISH analysis of somatic chromosomes. Except for CC+6R, all AMALs showed an entire (unrecombined) extrachromosome from A. roylei in the integral diploid background of A. cepa. A single recombination between A. cepa and A. roylei was observed on the extrachromosome in the remaining type. All alloplasmic AMALs possessing A. roylei cytoplasm showed high or complete pollen sterility. Only the autoplasmic CC+4R with A. cepa cytoplasm possessed relatively high pollen fertility. The bulbs of CC+4R displayed the distinct ovoid shape that discriminates them from spherical or oval ones in other AMALs. Downy mildew screening in the field showed higher resistance in A. roylei, a hypo-allotriploid (CCR-nR, 2n = 23), and an allotriploid (CCR, 2n = 24). Meanwhile, no complete resistance was found in some AMALs examined. This was the first trial toward the establishment of a complete set of A. cepa–A. roylei monosomic additions.     

Effects of sulphur nutrition on nitrogen and carbon metabolism in lucerne (Medicago sativa L.)

Studies were conducted with one-year-old plants of the perennial legume, lucerne ( Medicago sativa L.), to determine the effects of various levels of S (0.3, 1.5, and 7.5 m M ) on N₂-fixation, photosynthesis, herbage free amino acid pools and protein amino acids, levels of protein, and activities of key enzymes of leaf nitrogen and carbon metabolism. Sulphur deficiencies in the 0.3 m M S treatment, as determined by N:S ratios, did not appear until the second growth cycle. This treatment was severely S-deficient by the end of the third growth cycle. Sulphur deficiencies did not occur in the other two treatments over three cycles of growth. By the middle of the second growth cycle acetylene reduction rates of plants grown with 0.3 m M S were lower than those of other treatments. At the end of the third growth cycle acetylene reduction rates of the 0.3 m M S treatment were ca . 30–35% that of the other treatments. There was no effect of S-deficiency on photosynthesis, levels of leaf NADP-isocitrate dehydrogenase and glutamine synthetase activity, or concentrations of leaf protein over the course of the experiments. Levels of NAD-glutamate dehydrogenase, NAD-malate dehydrogenase, and glutamate oxaloacetate trans-aminase activity and concentrations of free methionine, arginine, urea, and ammonia increased, with S-deficiency. Boron concentrations were very high (ca. 300 μg/g dry wt.) in S-deficient plants. The mole percent methiomne in leaf protein decreased slightly and chlorophyll concentration decreased markedly with S-deficiency. This study suggests that N₂-fixation is affected early during S-deprivation of lucerne and that subsequent effects of S-deficiency may be due to loss of fixed nitrogen.     

Central cell nuclear-cytoplasmic incongruity:a mechanism for segregation distortion in advanced backcross and selfed generations of (Allium cepa L. x Allium fistulosum L.) x A. cepa interspecific hybrid derivatives

A model is presented as an explanation for an anomaly observed in germination and establishment and isozyme segregation patterns in Allium cepa x A. fistulosum F2BC3 populations generated in an introgression-breeding program. The F1BC3 parent of these populations was selected for its heterozygous PGI phenotype, Pgi-1(2/3); Pgi-1(2) was inherited from an A. cepa (Ac) seed parent and Pgi-1(3) from an A. fistulosum (Af) pollen parent. Germination and establishment was recorded for the F2BC3 progeny population. Segregation of Ac and Af Pgi-1 alleles was investigated in F2BC3 seeds and embryo and endosperm tissue was isolated and tested for isozyme expression. A pooled goodness-of-fit test of the segregation of Pgi-1 alleles in the populations to the expected Mendelian 1:2:1 ratio using the chi-square statistic gave a chi2 = 185.9, well beyond the accepted limits at 2 degrees of freedom. The 1:2:1 ratio expected for simple Mendelian inheritance was rejected, while a pooled chi-square goodness-of-fit test of the segregation of Pgi-1 alleles in the populations fit a 1:1 ratio with a chi2 = 0.203, based on the incongruity model. We present here the central cell nuclear-cytoplasmic incongruity hypothesis to explain the observed anomalies.     

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.     

GISH study of advanced generation of the interspecific hybrids between Allium cepa L. and Allium fistulosum L. with relative resistance to downy mildew

Genomic in situ hybridization (GISH) was used for a chromosomal composition study of the later generations of interspecific hybrids between A. cepa L. and A. fistulosum L., which are relatively resistant to downy mildew (peronosporosis). GISH revealed that F₂ hybrids, which did not produce seeds, were triploids (2n = 3x = 24) with 24 chromosomes and possessed in their complements 16 chromosomes of A. fistulosum L. and eight chromosomes of A. cepa L. or eight chromosomes of A. fistulosum L. and 16 chromosomes of A. cepa L. The advanced F₅ hybrid, which produced few seeds, was amphidiploid with 32 chromosomes. BC₁F₅ hybrid was triploid with eight chromosomes of A. fistulosum L. and 16 chromosomes of A. cepa L., which did not produce seeds. BC₂ (BC₁F₅) plant was amphidiploid that possessed 4 recombinant chromosomes and produced few seeds. GISH results point to 2n-gametes formation in macro- and microsporogenesis of the hybrids. The mechanism of 2n-gametes formation and the possibility of apomixes events in the backcrossing progeny are discussed.     

Improved method for regeneration and Agrobacterium-mediated transformation of Indian short-day onion (Allium cepa L.)

Plant Cell, Tissue and Organ Culture (PCTOC) - A high-auxin medium, usually used for callus induction, was not effective for Indian short-day onion cv. Bhima super. In this study, we found that the...     

Sulphur and nitrogen nutrition influence the response of chickpea seeds to an added, transgenic sink for organic sulphur

In order to increase the concentration of the nutritionally essential sulphur amino acids in seed protein, a transgene encoding a methionine- and cysteine-rich protein, sunflower seed albumin (SSA), was transferred to chickpeas (Cicer arietinum L). Transgenic seeds that accumulated SSA contained more methionine and less oxidized sulphur than the controls, suggesting that additional demand for sulphur amino acids from the expression of the transgene stimulated sulphur assimilation. In addition, the activity of trypsin inhibitors, a known family of endogenous, sulphur-rich chickpea seed proteins, was diminished in transgenic, SSA-containing seeds compared with the non-transgenic controls. Together, these results indicate that the reduced sulphur sequestered into SSA was supplied partly by additional sulphur assimilation in the developing transgenic seeds, and partly by some diversion of sulphur amino acids from endogenous seed proteins. Growth of chickpeas on nutrient with a high sulphur-to-nitrogen ratio increased the total seed sulphur content and the accumulation of sulphur amino acids in the seeds, and partly mitigated the effect of SSA accumulation on the trypsin inhibitor amount. The results suggest that free methionine and O-acetylserine (OAS) acted as signals that modulated chickpea seed protein composition in response to the variation in sulphur demand, as well as in response to variation in the nitrogen and sulphur status of the plant.     

The cytogenetic effects of Avenoxan on Allium cepa and its relation with pollen sterility

In the present study, the cytogenetic effects of the herbicide Avenoxan on meiotic chromosomes of Allium cepa and its relation with pollen sterility were studied. The bulbs with roots of Allium cepa were treated with a series of concentrations (0.1%, 0.2%, 0.4%) for 3, 6, 12 and 24 h. Controls and treated plants were shown to obtain M1 generation. All the used concentrations of the herbicide Avenoxan and exposure periods caused distinct increase in the number of abnormal cells when compared with the control. The type of the abnormalities induced: chromosome stickiness, bridges, laggards, univalents, quadrivalents and micronuclei. Avenoxan also caused pollen sterility. Increase of chromosomal aberrations was accompained by increase in pollen sterility.     

Identification of alliin,a constituent of Allium cepa with an inhibitory effect on platelet aggregation

A component of Allium cepa which inhibits platelet aggregation in vitro was isolated. The active compound was identified as alliin, (+)-S-allyl-L-cysteine sulfoxide. Alliin was synthesized and found to exert the same activity on platelet aggregation as the natural compound.     

Effect of different exposed lights on quercetin and quercetin glucoside content in onion (Allium cepa L.)

Quercetin and quercetin glucosides are the major flavonols present in onion (Allium cepa L.) and are predominantly present as quercetin, quercetin-3,4′-diglucoside and quercetin-4′-glucoside. Effect of different light wavelengths on onion after harvest and storage, with fluorescent, blue, red and ultra violet light influenced the quercetin and quercetin glucosides profile. In a peeled onion, all the light treatments elevated quercetin content in bulb. Among them, particularly fluorescent light effect was more eminent which stimulates the maximum synthesis of quercetin in onion. In case of whole onion bulb, skin and pulp showed different responses to light treatment, respectively. The pulp had the highest quercetin glucosides under blue light, whereas the lowest under fluorescent light. Onion skin showed nearly opposite pattern as compared to the pulp. In particular, light treatment proved to be a better way to increase the level of quercetin content in onions which might be utilized for industrial production of bioactive compounds from onion and onion waste products.     

The generation and consolidation of a radial array of cortical microtubules in developing guard cells of Allium cepa L.

The initiation and development of a radial array of microtubules (MTs) in guard cells of A. cepa was studied using immunofluorescence microscopy of tubulin in isolated epidermal layers. Soon after the completion of cytokinesis, MTs originate in the cortex adjacent to a central strip of the new, anticlinically oriented ventral wall separating the two guard cells. Cortical MTs extend from the mid-region of the central strip toward the cell edge where the ventral wall joins the inner periclinal wall. They then spread in a fan-like formation along the periclinal wall and gradually extend along the lateral and end walls as well. Many MTs criss-cross at various angles as they arc past the edge formed by the junction of the ventral and periclinal walls, but they do not terminate there, indicating that, contrary to previous report, the edge is not involved in MT initiation. Instead, the mid-region of the central strip appears to function as a planar MT-organizing zone. Initially, MTs radiate from this zone through the inner cytoplasm as well as the cortex. During cell expansion, however, the cortical MTs increasingly predominate and consolidate into relatively thick, long bundles, while the frequency of non-cortical MTs diminishes. The apparent density of MTs per unit surface area is maintained as the cells expand and gradually flex into an elliptical shape. The guard cells eventually separate completely at the pore site. The entire process is accomplished within about 12 h.Abbreviations DIC differential interference contrast - GC guard cell - MT microtubule To whom correspondence should be addressed.     

The effect of pre-incubation of Allium cepa L. roots in the ATH-rich extract on Pb uptake and localization

The positive influence of anthocyanin (ATH) on toxic metal-treated plant material is well documented; however, it is still not explained if it is caused by changes in element absorption and distribution. Therefore, detailed analysis of the effect of the ATH-rich extract from red cabbage leaves on Pb uptake and localization at morphological, anatomical and ultrastructural level was the goal of this study. Two-day-old adventitious roots of Allium cepa L. (cv. Polanowska) were treated for 2 h with the aqueous solution of Pb(NO₃)₂ at the concentration of 100 μM with or without preliminary incubation in the anthocyanin-rich extract from Brassica oleracea L. var. capitata rubra leaves (250 μM, 3 h). The red cabbage extract did not change the total Pb uptake but it enhanced the translocation of accumulated metal from roots to shoots. Within the pretreated roots, more Pb was deposited in their basal part and definitely smaller amount of the metal was bound in the apoplast of the outer layers of cortex cells. The ultrastructural analysis (transmission electron microscopy and X-ray microanalysis) revealed that the ATH-rich extract lowered the number of Pb deposits in intracellular spaces, cell wall and cytoplasm of root meristematic cells as well as in such organelles important to cell metabolism as mitochondria, plastids and nucleus. The Pb deposits were preferably localised in those vacuoles where ATH also occurred. This sequestration of Pb in vacuoles is probably responsible for reduction of metal cytotoxicity and consequently could lead to better plant growth.     

Effects of cycloheximide on preprophase bands and prophase spindles in onion (Allium cepa L.) root tip cells

Summary Effects of cycloheximide (CHM) on preprophase bands (PPBs) of microtubules (MTs) and on prophase spindle MTs in root tip cells of onion (Allium cepa L.) were examined. When root tip cells were treated with 36 M CHM for 0.5–4 h, the population of cells with a PPB did not decrease markedly although the population of mitotic cells and that of prophase cells with a PPB gradually decreased to half of the control root tips. In prophase cells treated with 11 and 36 M CHM for 2 h, the width of the PPB was 1.4 times broader than that in the prophase PPB without CHM. Electron microscopic observation on the cross section of the PPB showed that the number of MTs and the distance between adjacent MTs in prophase PPBs treated with CHM were similar to those in the early developmental stage of PPBs without CHM. The bipolar spindle, that appeared in late prophase was not seen in prophase cells treated with 11 M or higher concentrations of CHM for 2 h. In order to examine differences of perinuclear MT arrangement between CHM treated and non-treated prophase cells, arrangement of perinuclear MTs was examined by confocal laser scanning microscopy. In control cells without CHM, MTs appeared on the nuclear surface with several branched or cross over type MT foci in the cytoplasm when broad PPB formation started. These MT foci were replaced by the aster type MT foci, from which several MTs radiated along the nuclear surface. The aster type MT foci gradually gathered to form a bipolar spindle. MTs connecting the spindle pole region and the PPB were seen in late prophase. In CHM-treated cells (11-360 M for 2 h), branched and cross over type MT foci were prominent, even in prophase cells with well condensed chromosomes. Neither linkages of MTs between the spindle pole region and the PPB nor aster type MT foci were seen. These observations showed that CHM prevents the bundling of MTs in the PPB and also inhibits the formation of aster type MT foci that is essential for bipolar spindle development.     

Positive vs. negative interactions in Picris hieracioides L., a mid-successional species of Mediterranean secondary succession

A field experiment was designed to evaluate the importance offacilitative and competitive interactions in Picrishieracioides, a facultative biennial that colonises the early andthemid-stages of secondary succession in the Mediterranean region. Seedlings ofPicris hieracioides from populations of the early- (1year)and the mid-stages of field abandonment (15–40 years) were transplantedintwo adjacent old fields, abandoned for 4 (F4) and 20 years (F20) and thatdiffered markedly in floristic composition and vegetation structure. For twoyears, we experimentally manipulated competition (no-neighbours vs. naturalvegetation) and resource availability (addition of water and fertiliser vs.controls) in an attempt to evaluate their influence on survival, reproductivetiming, growth and reproductive output throughout the life cycle. Earlymortality was higher in non vegetated plots in both fields. Mortality ofseedlings was mainly due to herbivory by larvae of genusAgriotes. Flowering throughout the whole experiment wasalso facilitated by vegetation in the F4 field as a result of the positiveeffect of annual vegetation and remained unaffected in the F20 field because ofthe high competitive effect of established perennial vegetation. The additionofresources altered the effect of facilitation and competition on late seedlingsurvival. Survival was enhanced in the vegetated plots of the F4 field, becauseresource addition increased the shade provided by the canopy of vegetation,protected seedlings from temperature extremes and reduced water loss. Seedlingmortality also decreased in the F20 field but in a similar manner to vegetatedand non-vegetated subplots, and consequently the outcome of positive andnegative interactions remained neutral. The net effect of facilitation andcompetition resulted in interference later in the life cycle and appearedthrough final lower growth and reproduction in both fields. However, thegreatercompetition in 1994 than in 1995 in both fields, probably because the size ofthe rosettes, makes them less susceptible to competition, illustrates thedifficulty in predicting the outcome of competition solely of one season forfacultative biennial plants. The relative competition intensity calculatedusingonly survivors (RCI₁) was unaffected by habitat fertility in bothfields. In striking contrast, the relative competition intensity calculatedusing seedling mortality (RCI₂) was significantly higher in subplotswithout resource addition in both fields because of high seedling mortality invegetated subplots. Finally, there were no differences in the net effect offacilitation and interference processes among populations from early and midsuccessional stages showing that phenotypic plasticity buffers theenvironmentalselective pressures linked to successional processes.     

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.     

Effects of Photoperiod, Nitrogen Nutrition and Temperature on Inflorescence Initiation and Development in Onion (Allium cepa L.)

The effects of photoperiod, nitrogen nutrition and temperatureon inflorescence initiation and development in onion cv. Rijnsburgerand cv. Senshyu Semi-globe Yellow were studied in controlledenvironments. Rates of inflorescence initiation were estimatedusing the data for leaf numbers formed prior to flower formationand the rates of leaf initiation. At 9 °C inflorescenceinitiation was accelerated by long photoperiods particularlyfor cv. Rijnsburger where the average time for initiation was86 days in 8 h and 38 days in 20 h photoperiods. Initiationwas as rapid at 12 °C as at 9 °C but was slower at 6°C. A reduction in the nitrate concentration in the nutrientsolution from 0.012 to 0.0018 M greatly accelerated inflorescenceinitiation particularly in photoperiods and temperatures notconducive to rapid initiation. Cv. Senshyu initiated more slowlythan cv. Rijnsburger and was less sensitive to photoperiod andnitrogen level. The development rate of inflorescences afterinitiation was accelerated by long photoperiods and increasesin temperature from 6 to 12 °C but was retarded by the lowernitrogen level. Allium cepa L., onion, flower initiation, inflorescence development, photoperiod, nitrogen nutrition, temperature, vernalization