Determining relative abundance of specific DNA sequences in flow cytometrically sorted maize nuclei

A wide breadth of DNA content variation has been reported amongmaize lines. While the extent of this variation has been welldocumented, few studies have focused on its cause. Some of thenuclear DNA content variation has been explained by the presenceof B chromosomes or knobs. However, variation in these two structuresdoes not account for all of the observed variation. In orderto identify other fluctuating DNA sequences, a rapid and reliablemethod of estimating relative abundance of DNA sequences neededto be developed. The potential of flow cytometry in conjunctionwith spot hybridization for determining relative abundance ofspecific DNA sequences in maize was studied. Different numbersof G1 phase nuclei were sorted on nitrocellulose filters andnon-radioactive hybridization and signal detection performed.Results from these experiments revealed a significant, positivelinear correlation between the amount of target sequence andsignal density using both knob (R = 0.98) and ribosomal spacer(R =0.99) DNA sequences. In addition, G1 phase nuclei of eightinbred lines differing in the amount of knob heterochromatin,were sorted on to filters and the non-radioactive hybridizationand signal detection performed. A significant, positive linearcorrelation between C-band number and signal density (R =0.83;P = 0.0051) as well as between per cent heterochromatin andsignal density (R=0.96;P = 0.0002) was observed. These resultsindicate the usefulness of flow cytometry for spot hybridizationin determining the relative abundance of DNA sequences in themaize genome. Key words: Flow cytometry, copy number, non-isotope labelling, spot hybridization, flow sorting, Zea mays L.     

Flow Cytometric Determination of Relative Nuclear DNA Contents in Bicellulate and Tricellulate Pollen

Nuclear DNA content in mature pollen was measured with a flowcytometer Pollen of Lilium longiflorum, Dendranthema grandiflora(syn Chrysanthemum monfolium) and Zea mays was chopped and stainedwith the DNA fluorochrome DAPI DNA levels, expressed as arbitraryC values, were compared with those of nuclei isolated from leafor root material of the same plants In mature tricellulate pollen the generative cell is dividedafter second pollen mitosis into two sperm cells Tricellulatepollen from maize and chrysanthemum gave rise to one large 1Cpeak and, only in the case of chrysanthemum, a much smallerone at the 2C level These results suggest that the haploid nucleiof the vegetative as well as both sperm cells in tricellulatepollen are arrested in the G₁ stage of nuclear division Thesmall 2C peak in the case of chrysanthemum probably arose froma fraction of pollen with the sporophytic chromosome number(2n pollen) In contrast to this, mature bicellulate lily pollengave rise to two identical peaks at the 1C and the 2C levelFrom this result it was concluded that in bicellulate pollen,the 1C peak is caused by the signal of the haploid vegetativenucleus arrested in the G₁ stage of nuclear division, whereasthe 2C peak originates from the haploid generative nucleus whichhas already undergone DNA synthesis and is arrested in G₂ Lilium longiflorumThunb, lily, Dendranthema grandiflora Tzelev (syn Chrysanthemum morifolium Ramat ), chrysanthemum, Zea maysL, maize, male gametophytic cells, vegetative cells, generative cells, sperm cells, unreduced pollen, sporophytic cells, relative nuclear DNA contents, replication stage     

Flow cytometric and microscopic analysis of the effect of tannic acid on plant nuclei and estimation of DNA content

• Background and Aims Flow cytometry (FCM) is extensivelyused to estimate DNA ploidy and genome size in plants. In orderto determine nuclear DNA content, nuclei in suspension are stainedby a DNA-specific fluorochrome and fluorescence emission isquantified. Recent studies have shown that cytosolic compoundsmay interfere with binding of fluorochromes to DNA, leadingto flawed data. Tannic acid, a common phenolic compound, maybe responsible for some of the stoichiometric errors, especiallyin woody plants. In this study, the effect of tannic acid onestimation of nuclear DNA content was evaluated in Pisum sativumand Zea mays, which were chosen as model species. • Methods Nuclear suspensions were prepared from P. sativumleaf tissue using four different lysis buffers (Galbraith's,LB01, Otto's and Tris.MgCl₂). The suspensions were treated withtannic acid (TA) at 13 different initial concentrations rangingfrom 0·25 to 3·50 mg mL^–1. After propidiumiodide (PI) staining, samples were analysed using FCM. In additionto the measurement of nuclei fluorescence, light scatter propertieswere assessed. Subsequently, a single TA concentration was chosenfor each buffer and the effect of incubation time was assessed.Similar analyses were performed on liquid suspensions of P.sativum and Z. mays nuclei that were isolated, treated and analysedsimultaneously. FCM analyses were accompanied by microscopicobservations of nuclei suspensions. • Key Results TA affected PI fluorescence and light scatterproperties of plant nuclei, regardless of the isolation bufferused. The least pronounced effects of TA were observed in Tris.MgCl₂buffer. Samples obtained using Galbraith's and LB01 bufferswere the most affected by this compound. A newly described ‘tannicacid effect’ occurred immediately after the addition ofthe compound. With the exception of Otto's buffer, nuclei ofP. sativum and Z. mays were affected differently, with pea nucleiexhibiting a greater decrease in fluorescence intensity. • Conclusions A negative effect of a secondary metabolite,TA, on estimation of nuclear DNA content is described and recommendationsfor minimizing the effect of cytosolic compounds are presented.Alteration in light scattering properties of isolated nucleican be used as an indicator of the presence of TA, which maycause stoichiometric errors in nuclei staining using a DNA intercalator,PI.     

Limited Genome Size Variation in Sesleria albicans

The extent and significance of intraspecific genome size variationin plants continues to be a matter of discussion: in some speciesconsiderable variation has been described, while no variationhas been detected in other taxa. In the present study, intraspecificgenome size variation was analysed in a perennial grass Sesleriaalbicans Kit. ex Schult. (Poaceae). Flow cytometry was usedfor the analysis of nuclear DNA content in ten geographicallyisolated populations ofS. albicans . Despite long-term isolationand lack of gene-flow between the populations, only negligibleinter-population differences were found. Although the differencesbetween the populations were statistically significant, themaximum inter-population difference reached only 1.6% of themean 2C value (9.78 ± 0.04 pg). The variation was notcorrelated with geographical location or with altitude of thepopulations analysed. The present study clearly demonstratesthat S. albicans belongs to the plant taxa with a highly stablegenome size. Copyright 2000 Annals of Botany Company Sesleria albicans, genome size, nuclear DNA content, intraspecific variation, flow cytometry, Europe     

Comparison of [14C]oryzalin Uptake in Root Segments of a Sensitive and a Resistant Species

Uptake of the dinitroaniline herbicide oryzalin (3,5-dinitro-N⁴,N⁴-dipropylsulfamlamide) and its effect on root growth werestudied using 5 mm corn (Zea mays L.) and pea (Pisum sativum)root apices. Pea root growth was much less susceptible to oryzalinthan corn root growth. Uptake studies showed that pea root apicesalso accumulated much less [¹⁴C]oryzalin and had a lower bindingaffinity for this herbicide. [¹⁴C]oryzalin was not metabolizedin root apices from either species. Thus, the differential susceptibilityto oryzalin in the case of corn versus pea can be explained,at least in part, by differences in oryzalin uptake and accumulationby roots. Oryzalin, dinitroaniline herbicides, Zea mays, Pisum sativum     

Separation of o-Phthaldialdehyde Derivatives of Free Amino Acids from Plant Tissues by Isocratic Reverse-phase High-performance Liquid Chromatography

A reverse-phase high-performance liquid chromatography procedurehas been developed for rapid separation and quantitation offree amino acids as o-phthaldialdehyde derivatives. A two stepisocratic solvent system was used which enabled an accurateanalysis at nanomole level. However, two major disadvantagesto this procedure were the lack of reaction of proline and theco-elutions of threonine/glycine and tryptophan/methionine.The free amino acids in Zea mays roots were separated by usingthe method described. Amino acids, liquid chromatography, o-phthaldialdehyde derivatives, reverse-phase chromatography, Zea mays L, maize, corn     

Submicroscopic Structure of the Cereal Starch Grain

The submicroscopic structure of the starch grains of Zea maysand Triticum sativum was studied electron-optically from replicasmade of internal, fracture surfaces. In corn starch, long cylindricalmicrofibrils were found, arranged in a radial direction andimbedded in an amorphous matrix. Their diameter was uniformlyabout 200 Å. Microfibrils were also indicated in wheatstarch because of the prominence of their ends in surface view.Many microfibrils in corn starch appeared to be helically coiled.The more ordered starch substance was presumed to be localizedprincipally in the microfibrils.     

Biosynthesis of Cytokinin in Germinating Seeds of Zea mays

The embryos of germinating Zea mays seed were supplied with[¹⁴C]-adenine Following incubation, the tissue was extractedand extensively purified by non-exchange chromatography andthin layer chromatography. Radioactivity was found to be incorporatedinto zeatin nucleotide indicating that the embryo in the germinatingseed is capable of cytokinin biosynthesis. Key words: Zea mays cytokinin, zeatin nucleotide, biosynthesis, seed     

Intraplant Nuclear DNA Content Variation in Diploid Nuclei of Maize (Zea mays L.)

Diploid nuclei from stem, mesocotyl, nodal root and root tiptissue of two maize hybrids were examined with respect to theirDNA content. The nuclei were isolated and stained with DAPIand passed through a flow cytometer-cell sorter. The titrationcurve for each tissue was determined. Significant variationwas observed among nuclei of different tissue types. Stem androot tips had the highest diploid nuclear DNA amounts while2-week-old mesocotyl had the lowest diploid nuclear DNA amount.These results provide evidence that during plant developmentand differentiation, the amount of DNA within a diploid nucleuschanges through loss of specific DNA sequences. This study alsodemonstrates the sensitivity of flow cytometry in detectingsmall intraplant variation in nuclear DNA. Key words: Flow cytometry, fluorochrome DAPI, DNA content, tissue differentiation, plant development     

Microsporogenesis and the Mechanism of Cytoplasmic Male Sterility in Maize

Examination of the structural changes occurring during microsporogenesisin cms-T, -C and -S Zea mays L. with different nuclear backgroundsrevealed considerable variations in the stages at which abortionof the sporocytes occurred within each cytoplasmic genotype.This variation was not associated with the nuclear background.Significantly, we found that the mitochondria in cms-T plantsdid not always follow the pattern of degeneration describedpreviously. We conclude that these observations are not consistentwith a proposed mechanism of cytoplasmic male sterility involvingthe direct, antagonistic action of an anther produced substanceon an altered mitochondrion or plastid, that is functionallynormal in the rest of the plant. The value of microscopic studiesin detecting variation in apparently uniform material is emphasised,though it is recognized that other methods will be requiredto elucidate the mechanism of male sterility. male sterility, mitochondria, cytoplasmic inhertance, microsporogenesis, Zeamays L., maize, corn     

Silica and Ash Content and Depositional Patterns in Tissues of Mature Zea mays L. Plants

Ash and silica content and their depositional patterns in differenttissues of the mature corn plant (Zea mays L.) were determined.Ash and silica were highest in the leaf blades (up to 16.6 and10.9 per cent, respectively) followed by the leaf sheath, tassel,roots, stem epidermis and pith, and ear husk. The percentageof ash as silica was also highest in the leaves. Silica wasextremely low in the kernels. The upper stem epidermis and pithcontained nearly twice the silica content as did the lower portion.The patterns of ash and silica distribution were similar inplants grown in two different areas of Kansas, but were in lowerconcentration in the leaves and leaf sheaths from the area withlower soluble silica in the soil. Silica was deposited in theepidermis in a continuous matrix with cell walls showing serratedinterlocking margins in both leaves and stem. Rows of lobedphytoliths of denser silica were found in the epidermis as wellas highly silicified guard cells and trichomes. The silica matrixof the epidermis appears smooth on the outer surface and porousor spongy on the inner surface. Zea mays L. Corn, maize, ash content, silica deposition, scanning electron microscopy     

A Lack of Nuclear DNA Content Variability Among Wheat Near Isolines Differing in Aluminium Response

Nucleotypic variation has been speculated to play a role inthe adaptation of crop species to environmental stress. Theobjective of this study was to determine if nuclear DNA contentvariability was associated with aluminium (Al) tolerance inwheat. Six wheat (Triticum aestivumL.) near isolines (differingin Al response), two recurrent parents (Al sensitive), and onedonor parent (Al tolerant) were all analysed for nuclear DNAcontent using flow cytometry. A 1.7% variation in nuclear DNAcontent was observed among the nine wheat lines. No associationbetween Al response and nuclear DNA content was observed. Allof the wheat near isolines had a nuclear DNA content similarto their recurrent parent. The wheat genome appears to be stablewith no unusual inheritance of nuclear DNA content observed.Flow cytometric analysis proved to be sensitive enough to detectnuclear DNA content variability at the level of 0.5% variationamong wheat lines.Copyright 1999 Annals of Botany Company Genome size,Triticum,breeding, near isogenic.     

Reference standards for determination of DNA content of plant nuclei

Flow cytometry was used to compare 14 potential reference standards for plant DNA content determination. Both chicken and plant internal standards were used, as were propidium iodide (PI) and 4'-6-diamidino-2-phenylindole (DAPI) as fluorochromes. Means and standard errors of the means are presented for the 14 potential reference standards, and the means are compared to those obtained by Feulgen densitometry. Five species are recommended as an initial set of international standards for future plant DNA content determinations: Sorghum bicolor cv. Pioneer 8695 (2C = 1.74 pg), Pisum sativum cv. Minerva Maple (2C = 9.56 pg), Hordeum vulgare cv. Sultan (2C = 11.12 pg), Vicia faba (2C = 26.66 pg), and Allium cepa cv. Ailsa Craig (2C = 33.55 pg). It is recommended that the reference standard of choice be one with 2C and 4C nuclear DNA content peaks similar to, but not overlapping, the 2C and 4C peaks of the target species. We recommend PI as the fluorochrome of choice for flow cytometric determination of plant DNA content. DAPI should be used only if the estimated DNA value is corroborated by using a second stain that has no bias for AT- or GC-rich sequences within genomes.     

High-resolution flow cytometry of nuclear DNA in higher plants

Summary High-resolution flow cytometry of nuclear DNA in higher plants has been performed from chopped plant tissues and plant protoplasts. A preparation and staining procedure with the DNA specific fluorochrome DAPI, successfully employed for precise flow cytometric DNA analysis of animal and human cells has been used in a slightly modified manner for the DNA analysis of plant cell material. High-resolution DNA histograms coefficients of variation about 1–1.5% have been obtained routinely from plant species with different DNA content. Staining of nuclei with DAPI in combination with the protein fluorochrome sulforhodamine 101 allows bi-parametric analysis of nuclear DNA and protein. The described simple and precise method might be very promising for the analysis of DNA in basic and applied cytogenetic investigations of plant cell research.Abbreviations CV coefficient of variation - DAPI 4,6-diamidino-2-phenylindole - SR 101 sulforhodamine 101     

Genome size variation and C-band polymorphism inAlstroemeria aurea, A. ligtu andA. magnifica (Alstroemeriaceae)

Among a total of 43 accessions ofAlstroemeria aurea, A. ligtu andA. magnifica nuclear DNA amounts (2C-values) showed significant intraspecific variation, 1.09, 1.21 and 1.15 fold, respectively, when determined through flow cytometric measurements of fluorescence of propidium iodide (PI) stained nuclei. After staining with another fluorochrome, 4,6-diamidino-2-phenylindole (DAPI), an intraspecific variation of 1.10, 1.11 and 1.12 fold, respectively, was found. C-band polymorphisms were present among and within the accessions of all three species. In some cases only very small differences in C-banding pattern were observed. In other cases, however, differences were more prominent. Besides C-band polymorphism, there were also instances of chromosome length polymorphism, which concerned the total chromosome complement or single chromosomes. The variation in nuclear DNA amount inA. aurea andA. ligtu was more or less continuous, except for one accession ofA. ligtu subsp.simsii. Artificial selection and possibly introgression of chromosomes from other species may have moulded the karyotypes of some of the accessions ofA. aurea, a species that has been under cultivation for more than 160 years. The variation as observed inA. magnifica subsp.magnifica was discontinuous and could be due to a broad species concept.     

The Involvement of Glucose-6-Phosphatase in Mucilage Secretion by Root Cap Cells of Zea mays

In order to determine the involvement of glucose-6-phosphatasein mucilage secretion by root cap cells, we have cytochemicallylocalized the enzyme in columella and peripheral cells of rootcaps of Zea mays. Glucose-6-phosphatase is associated with theplasmalemma and cell wall of columella cells. As columella cellsdifferentiate into peripheral cells and begin to produce andsecrete mucilage, glucose-6-phosphatase staining intensifiesand becomes associated with the mucilage and, to a lesser extent,the cell wall. Cells being sloughed from the cap are characterizedby glucose-6-phosphatase staining being associated with thevacuole and plasmalemma. These changes in enzyme localizationduring cellular differentiation in root caps suggest that glucose-6-phosphataseis involved in the production and/or secretion of mucilage byperipheral cells of Z. mays. Zea mays, corn, glucose-6-phosphatase, columella cell, peripheral cell, mucilage, secretion, cytochemistry     

Changes in chloroplast DNA during development in tobacco, Medicago truncatula, pea, and maize

We examined the DNA from chloroplasts obtained from young and fully expanded leaves of tobacco (Nicotiana tabacum L.), Medicago truncatula, pea (Pisum sativum L.), and maize (Zea mays L.). The changes in plastid DNA content and structure were monitored by four independent methods: 4′,6-diamidino-2-phenylindole (DAPI) staining with intact chloroplasts, in situ DAPI staining of cytological sections, ethidium bromide staining at the single-molecule level after exhaustive deproteinization of lysed chloroplasts, and pulsed-field gel electrophoresis. During leaf development, we found a decline of chloroplast DNA (cpDNA) in all four plants. For tobacco, for which plants can readily be regenerated from somatic cells, cpDNA persisted longer than in the other three plants. We also found a striking progression from complex multigenomic DNA molecules to simple subgenomic molecules during plastid development. Although the decrease in molecular size and complexity paralleled the decrease in DNA content per plastid, 6% of the chloroplasts in a fully expanded tobacco leaf still contained DNA in complex branched structure, whereas no such complex structures were found in mature leaves for the hard-to-regenerate maize.     

Substantial Genome Size Variation in Taraxacum stenocephalum (Asteraceae, Lactuceae)

There are only a few exceptions to the rule that polyploidy in Taraxacum is associated with agamospermy. One of them is the sexual, tetraploid species Taraxacum stenocephalum. Incidentally, remarkable variation in karyology was found in this species. The present study aims to confirm this variation by an extensive screen of nuclear DNA content. Individuals from two large populations in the Lesser and Greater Caucasus, Georgia were analyzed using flow cytometry to ascertain intraspecific nuclear DNA content variation. Across the whole data set comprising all 159 individuals, a 1.223-fold difference was detected based on propidium iodide (PI) analyses. To verify this finding, we compared flow-cytometric data obtained using DAPI (4′,6-diamidino-2-phenylindole) and PI staining using a representative subset of individuals. This comparison revealed a 1.194-fold difference in DNA content for DAPI and a 1.219-fold difference for PI. Mean nuclear genome size in absolute terms (2C value ± s.d.) was estimated at 4.38?±?0.21 pg, ranging from 4.01 pg to 4.89 pg, despite the invariable chromosome counts of 2n?=?32. A regression analysis comparing the datasets for DAPI and PI staining found a strong correlation between data obtained by the DAPI and PI dyes (R?=?0.976; P?=?0.0001). Simultaneous high-resolution flow-cytometric analyses also proved the accuracy of our findings. We discuss possible sources of these large differences in DNA content within Taraxacum stenocephalum. Further research is needed to identify the source of this remarkable variation.     

Changes in Lipids of Bean Seeds (Phaseolus vulgaris) and Corn Caryopses (Zea mays) Aged in Contrasting Environments

Seeds of French bean (Phaseolus vulgaris L. cv. The Prince)and caryopses of sweet corn (maize; Zea mays L.F1 hybrid Firstof All) were stored in environments of 79% relative humidityand 25 °C, 80% r.h. and 40 °C or 100% r.h. and 45 °C,giving ageing (loss of gcrminability and vigour) over periodsof months, weeks or days, respectively. The relationship betweenchanges in lipids and changes in germinability or vigour wasunaffected in general by the speed of ageing. In the corn caryopsesthere was no evidence of hydrolysis of phospholipids or peroxidationof fatty acids during ageing. In the bean seeds phosphatidicacid increased during the ageing period and phosphatyl cholinedeclined. The percentage of fatty acids as hnolenic acid initiallyfell during bean ageing, but in the slower ageing conditionsit rose again as germination reached zero. In bean, the presenceof phosphatidic acid could be a sensitive indictator of lossof vigour, but relative proportions of the different fatty acidswould be a misleading indicator of quality. Rapid artificialageing may be an adequate model, in some species, of ageingat moderate speeds and of ageing under some ambient conditions. French bean (Phaseolus vulgaris L. cv. The Prince), sweet corn (maize, Zea mays L., Fl hybrid First of All), seed ageing, phospholipids, fatty acids