Residual Nuclear structures fromZea mays L.

Nuclei fromZea mays L. root tip meristematic cells were treated according to the conventional method for nuclear matrix isolation and according to a recently adapted procedure for isolation of nuclear shells from plant cells. In the first case, after high salt extraction of proteins and DNase I and RNase digestions, residual structures are obtained consisting of a periferal layer and an internal network. The obtained structures are very similar to the nuclear matrix preparations from animal cells. In case nuclei are swollen in EDTA first, digested with DNase II and RNase prior high salt treatment, structures devoid of internal network are obtained. The analogous residual structures were shown forPhaseolus vulgaris L. meristematic root cells nuclei (Galcheva-Gargovaet al. 1988). The morphology and the protein composition of the two types of residual structures suggest that the organization of scaffold structures from plant nuclei is very similar to the one from animal cell nuclei.     

The Plant Nucleoskeleton: Ultrastructural Organization and Identification of NuMA Homologues in the Nuclear Matrix and Mitotic Spindle of Plant Cells

In the present work we investigate the structural organization of the nucleoskeleton ofAllium cepameristematic root cells. Resinless sections reveal for the first time a residual filamentous network in plant nuclei. This network is composed of branched knobbed filaments with associated globular structures, connected to the lamina and to the dense aggregates of different sizes. Results of immunoblotting show that many components of this network are homologues of intermediate filament-type proteins. NuMA, a coiled-coil protein related to intermediate filaments, found in animal cells, can also be detected in this plant nuclear matrix system. Immunofluorescence reveals a diffuse distribution of the animal NuMA homologues in plant nuclear core filaments in interphase. Resinless immunoelectron microscopy further reveals a distribution along the extended filaments and the dense aggregates. During mitosis, in contrast to the accumulation at the poles in animal cells, NuMA homologues in plant onion cells show a diffuse pattern, which may correspond to the spindle matrix. Our data are the first report of the conservation in plants of NuMA proteins, which may be involved in both nuclear and mitotic spindle organizations.     

Attachment of DNA to nucleolar and nuclear skeletal structures as visualized by Kleinschmidt molecular spreading

Co-isolated residual nuclear shells and residual nucleoli from membrane-depleted rat liver nuclei were spread according to Kleinschmidt's method. Comparison of the spread residual structures isolated from nuclear shells and spread pore complex-lamina isolated from nuclear envelopes showed that these residual structures are morphologically identical. Furthermore, our nuclear shell isolation procedure allowed visualization of DNA strands bound to a granular component of the lamina. The fragmentation of nuclear shells allowed us to obtain well-spread nucleolar remnants, in which we observed DNA strands anchored on a residual nucleolar network attached to the lamina. The different molecular features revealed by the spreading of residual nucleolar structures suggest that both non-transcribing nucleolar DNA and active ribosomal genes are linked to the nucleolar network. Although the exact nature of this network remains to be defined, the results of the present study strongly suggest that the DNA molecules of the chromosomes bearing ribosomal genes have many sites of attachment to a non-chromatin nucleolar network which can be referred to as a nucleolar skeletal complex.     

Maize calreticulin localizes preferentially to plasmodesmata in root apex

Using a polyclonal antibody raised against calreticulin purified and sequenced from maize, we performed an immunocytological study to characterize putative domain-specific subcellular distributions of endoplasmic reticulum (ER)-resident calreticulin in meristematic cells of maize root tip. At the light microscopy level, calreticulin was immunolocalized preferentially at cellular peripheries, in addition to nuclear envelopes and cytoplasmic structures. Punctate labelling at the longitudinal walls and continuous labelling at the transverse walls was characteristic. Immunogold electron microscopy revealed plasmodesmata as the most prominently labelled cell periphery structure. In order to further probe the ER-domain-specific distribution of maize calreticulin at plasmodesmata, root apices were exposed to mannitol-induced osmotic stress. Plasmolysis was associated with prominent accumulations of calreticulin at callose-enriched plasmodesmata and pit fields while the contracting protoplasts were depleted of calreticulin. In contrast, other ER-resident proteins recognized by HDEL peptide and BiP antibodies localized exclusively to contracted protoplasts. This finding reveals that, in plasmolysed cells, calreticulin enriched ER domains at plasmodesmata and pit fields are depleted of other ER-resident proteins containing the HDEL retention peptide.     

Heat-induced morphological and biochemical changes in the nuclear lamina from Ehrlich ascites tumor cells in vivo

Membrane-depleted nuclei from Ehrlich ascites tumor (EAT) cells isolated at low ionic strength in the presence of EDTA exhibit highly decondensed chromatin fibers and a loss of morphologically identifiable nucleoli. Treatment of these nuclei with nucleases and 2 M NaCl followed by low-speed centrifugation permitted the facile isolation of the nuclear lamina layer. Under the same conditions, but after heat-shock treatment of the living cells, the chromatin appears in a more condensed state, the nucleoli are well-defined, and the nuclear lamina layer was destabilized in concert with the appearance of an internal nuclear matrix and nucleolar skeleton. Furthermore, we also found both an increase in the protein mass as well as the appearance of a relatively large number of new proteins in this fraction, which are phosphorylated. The major proteins of the nuclear lamina, the lamins, and the residual vimentin remained insoluble. These heat-shock-induced changes were also accompanied by a dephosphorylation of lamins A and C but not of lamin B.     

Acid-soluble chromosomal proteins in maize root and callus cells and after rhizogenesis induction in callus tissues

Callus tissues originating fromZea mays root meristem, induced for rhizogenesis callus, meristematic and differentiated maize root cells for isolation of nuclei and acid-soluble chromosomal proteins were used. Cytological investigations proved that rhizogenesis begins with the formation of meristematic centres, followed by root differentiation about 5–12 days after the treatment with α-naphtalene acetic acid (NAA). When applying electrophoresis in 15% polyacrylamide gel, differences between the electrophoretic profiles of acid-soluble chromosomal proteins, isolated from root cells and from callus tissues, were established. The main differences concern histone H1 and probably H4. There are no differences between electrophoretic patterns of acid-soluble chromosomal proteins of nonorganized callus and callus induced for rhizogenesis. The possible explanation of these results is discussed.     

Isolation and characterization of a functional A-type cyclin from maize

Cyclins are involved in the regulation of cell cycle progression in eukaryotes. We have isolated a cyclin cDNA clone, cycZm2w, from maize root tip cells, which fits best into group A2 of current plant cyclin gene classification schemes. The cDNA encodes a protein with a domain homologous to the cyclin box of mitotic cyclins. Complementation studies revealed that cycZm2w was able to rescue a budding yeast cyclin-deficient mutant (BF305–15d#21). As expected, cycZm2w is expressed in organs of the maize plant that possess meristematic activity, but is especially prominent in the proliferating regions of the root apex.     

Interphase nuclear structure and heterochromatin in Phaseolus plant species

Interphase nuclear structure was studied in five Phaseolus plant species. All the species showed chromocentric nuclear organization in both meristematic and differentiated cells. The number of chromocenters appeared to be a species-specific character. Percentage heterochromatin values, as determined by using three different staining techniques, were higher in meristematic cells than those in differentiated cells. There was a close correspondence between percentage heterochromatin and amount of highly repetitive DNA implying a possible involvement of the latter in chromatin condensation.NCL Communication No. 3381To whom all the correspondence should be addressed.     

Correlation of reduced chilling injury with Increased spermine and spermidine levels in zucchini squash

By means of a biparametric cytofluorimetric analysis it is possible to distribute meristematic plant cells in a variety of cell cycle sub-compartments, unidentifiable by DNA measurements alone. In this work, an asynchronous proliferating cell population of pea root meristems was divided into different sub-compartments of the cell cycle phases, i.e. G1A, G1B, S. G2A and G2B on the basis of their DNA-nuclear protein content. By means of the same biparametric analysis, differentiated mesophyll cells and quiescent cells of embryo roots, indicated as G0 and G2Q, were distinguished from cycling cells by their low nuclear protein content. These results conform to those of some analyses performed on animal cells in culture and show that it is possible to get a major insight into cell cycle kinetics and its control in a natural system such as root meristem.     

Overexpression of the lamina proteins Lamin and Kugelkern induces specific ultrastructural alterations in the morphology of the nuclear envelope of intestinal stem cells and enterocytes

The nuclear envelope has a stereotypic morphology consisting of a flat double layer of the inner and outer nuclear membrane, with interspersed nuclear pores. Underlying and tightly linked to the inner nuclear membrane is the nuclear lamina, a proteinous layer of intermediate filament proteins and associated proteins. Physiological, experimental or pathological alterations in the constitution of the lamina lead to changes in nuclear morphology, such as blebs and lobulations. It has so far remained unclear whether the morphological changes depend on the differentiation state and the specific lamina protein. Here we analysed the ultrastructural morphology of the nuclear envelope in intestinal stem cells and differentiated enterocytes in adult Drosophila flies, in which the proteins Lam, Kugelkern or a farnesylated variant of LamC were overexpressed. Surprisingly, we detected distinct morphological features specific for the respective protein. Lam induced envelopes with multiple layers of membrane and lamina, surrounding the whole nucleus whereas farnesylated LamC induced the formation of a thick fibrillary lamina. In contrast, Kugelkern induced single-layered and double-layered intranuclear membrane structures, which are likely be derived from infoldings of the inner nuclear membrane or of the double layer of the envelope.     

Underrepresentation of nuclear DNA sequences in differentiating root cells ofVicia faba

Summary Data from cytological and biochemical analyses are presented on the behaviour of nuclear DNA during the differentiation ofVicia faba root cells. From the terminal 10.5 mm of the root, three segments were dissected by cutting transversely the root at 0.5 (segments I, meristematic cells), 4.5 (segment II, both meristematic and differentiating cells) and 10.5 mm (segment III, differentiating and/or differentiated cells) from the tip. Cytophotometric determinations of Feulgen absorptions in cell nuclei of the three root segments, carried out in preparations subjected to hydrolysis curve, revealed a lesser amount of nuclear DNA in differentiating cells when compared to the meristematic ones. Analyses of the reassociation kinetics of the DNAs extracted separately from the three root segments showed differences in the frequency of highly repeated sequences, which amount to 11.0, 8.6, and 7.5% of the total DNA in segments I, II, and III, respectively. Density gradient centrifugations in CsCl revealed a lighter satellite in the DNAs from segments I and II (ca. 5.4 and 3.8% of the total DNA, respectively) and no satellite in the DNA from segment III. It is suggested that underrepresentation of repeated DNA sequences occurs in differentiating cells and is a determining factor of the discharge of a cell from the mitotic activity.     

Changes in nuclear and nucleolar protein content during the growth and differentiation of root parenchyma cells in plant species with different DNA-endoreplication dynamics

Using cytophotometric procedures, we measured the nuclear and nucleolar protein content of successive zones of growth and differentiation in consecutive (1-7 mm) root segments obtained from eight species of the Angiospermae after staining the preparations with Feulgen-Naphthol Yellow S (F-NYS). In meristematic cells the nuclear and nucleolar protein content was found to double during the cell cycle. In species in which differentiation occurs at the same time as nuclear DNA endoreplication, i.e. Vicia faba subsp. minor, V. faba subsp. major, Pisum sativum, Hordeum vulgare and Amaryllis belladonna, the pool of nuclear proteins observed during the G2 phase of the cell cycle was seen in the differentiated zone in nuclei containing 8C DNA. Species in which differentiation is not accompanied by the process of nuclear DNA endoreplication, i.e. Levisticum officinale, Tulipa kaufmanniana and Haemanthus katharinae, exhibited the highest nuclear proteins content during the G2 phase of the cell cycle; comparably high values were not found in the differentiated zone. A decrease in nucleolar protein content was observed during the process of differentiation, this tendency being more evident in the studied species that do not exhibit endoreplication.     

Cytohistological Studies on the Tissue Culture of Olea europaea L. II. Histogenesis and Organogenesis

After the calli derived from Olea europaea stem tissue have been introduced to the subculture on the solid medium for differentiation, a periderm was partially formed from the wound cambium in the outer region of the callus. At the same time, some scattered tracheids and vascular bundles were differentiated in the inside of the callus. These vascular bundles did not form a vascular system and also had no rela- tion to the organogenesis. In addition, there were some embryonic cells induced at random from parenchymatous cells in the callus, and these embryonic cells were characterized as the meristematic cells. Two types of meristematic tissues, namely, meristematic cellular mass and meristematic nodule, were produced by different types of mitotic division respectively. The meristematic nodules formed a growth center without any differentiation, but later, they were differentiated tracheids in the inner surrounded by the cambium-like cells. With monopolarity the root primordia were produced from this type of nodules. But the adventitious buds were derived from the meristematic cellular masses. Therefore, realize that the process of differentiation and dedifferentiation all occurs in the callus tissue. The structural differences among the nodules with tracheids, and the origin of buds and root primordia are also briefly discussed.     

Image Analysis of Maize Root Caps--Estimating Cell Numbers from 2-D Longitudinal Sections

The cap of the primary root of maize produces several thousandborder cells that are shed from the outside of the cap eachday. Border cell production is important in the penetrationof soil by roots, and in influencing the activity of both beneficialand pathogenic organisms in the rhizosphere. To improve understandingof the dynamics of border cell production, it is desirable toknow the number of cells in different parts of the root cap.An image analysis procedure was used to quantify cell dimensionsand locations in the median longitudinal section of maize (Zeamays L.) root caps. Calculations based on root symmetry werethen used to estimate the number of cells in 3-dimensions. Ourestimation procedure was tested initially using regular arraysof identical square and hexagonal shapes to represent cells.It was then tested using two different tissues showing analogousarrays: a transverse section through the maize root cap junction,and a transverse section through a barley root. Good linearcorrelations were obtained between the number of cells estimatedand the number of cells actually counted in the microscope.The numbers of cells in the whole maize root cap (8870 ±390) were then estimated from longitudinal sections. These numbersof cap cells agreed with values that had been estimated formaize by other methods. In the first tier of the cap meristem,ten-times more meristematic cells were located in the cap flanks(>500 cells) than were present in the columella portion.Similarly, only 7% of cells in the outermost layer of the rootwere associated with the columella region of the cap, a fractionwhich compared well with previous measurements of sloughed cellsextracted from rhizosphere sand. This present technique canbe applied to estimate the numbers of cells in any cylindricallysymmetrical tissue from two-dimensional sections. Copyright2001 Annals of Botany Company Anatomy, border cells, cell production, image analysis, maize, rhizosphere, root cap, sloughing, stereology, Zea mays L     

Identification of specific plant nucleolar phosphoproteins in a functional proteomic analysis

The soluble fraction of nuclear proteins is a functionally significant fraction, since it has been shown that it contains ribonucleoproteins active in nuclear RNA metabolism. The aim of this work was to detect variations associated with cell proliferation, by comparing two-dimensional proteomes obtained from the soluble fractions of onion nuclei isolated from actively proliferating root meristematic cells versus nonmeristematic root cells. In particular, we have studied the physicochemical features of the major nucleolar protein NopA100, a highly phosphorylated, nucleolin-like protein. A total of 384 spots were quantified in meristematic nuclei, while only 209 were detected in nonmeristematic nuclei. The comparison of both proteomes resulted in the determination of specific spots for each proliferative state and those which were common to both cases. Furthermore, among these latter, we could discriminate quantitative differences. Interestingly, well-known nucleolar proteins, such as RNA polymerase I, B23 and the nucleolin-like protein NopA100, were significantly increased in proliferating cells. Western blots with anti-NopA100 antibody demonstrated 26 spots in the meristematic sample. All the spots detected were clustered at 100 kDa and were distributed through an isoelectric point (pI) range of 4.3-6.6. In contrast, only seven spots were found in the extract from nonmeristematic nuclei, and the pI range was shortened to 4.8-6.1. These results indicate that the state of NopA100 phosphorylation correlates with the degree of nucleolar activity, i.e. the protein is more highly phosphorylated in cycling cells. We have also analyzed the bidimensional silver staining of the nucleolar organizing region (Ag-NOR) pattern of the soluble nuclear fraction in order to identify plant cell phosphoproteins that are considered to be markers of proliferation. These experiments demonstrated that NopA100, the onion, nucleolin-like protein, is an Ag-NOR protein. In addition we found that the plant homologue of the vertebrate nucleolar phosphoprotein B23 migrated as two clusters of acidic spots, 43 and 42 kDa respectively in molecular mass. The differences between these features and those described for mammalian cells is discussed. Our results demonstrate that the use of protein fractionation procedures with functional significance and the location of candidate spots by indirect techniques are advantageous, complementary methods to random selection procedures for proteomic studies involving further mass spectrometry analysis.     

Anchorage of the nucleolus in the pore complex-lamina by a DNA-bearing structure masked in situ in rat liver nuclei

We have developed a method by which nuclear shells containing nucleoli can be isolated from membrane-depleted rat liver nuclei. This method involves the removal of the internal chromatin. This chromatin is expelled from the nuclear shell using combinations of low and high ionic strength buffers. The expelled internal part is subsequently digested with DNase I or micrococcal nuclease. Examination by electron microscopy of the nuclear and the nucleolar structures at various steps of the isolation procedure shows that the nucleoli are anchored in the peripheral lamina by a pedicle that is continuous with an intranucleolar network. This network is masked in situ by nucleolar granules. The pedicle and the network which support the nucleolar DNA are composed mainly of non-histone proteins insoluble in 2M NaCl.     

Isolation and characterization of nuclear lamina from Ehrlich ascites tumor cells

We have developed a simple and rapid method for isolation of purified nuclear lamina from Ehrlich ascites tumor cells. The procedure employs chromatin structures prepared from whole cells at low ionic strength and is carried out under conditions that minimize the formation of artifactual protein-DNA complexes. When the isolation is performed in the presence of EDTA, nuclear lamina without distinct pore complexes is obtained. In the absence of EDTA, intact pore complexes and a large amount of vimentin 100 A filaments are seen associated with nuclear lamina. The main nuclear lamina proteins are characterized using gel electrophoresis, immunoblotting, and two-dimensional peptide mapping. An extensive structural homology is found between lamin A and lamin C, whose peptide maps differ by only one major spot, whereas lamin B has apparently unrelated pattern.     

Anchorage of the nucleolus in the pore complex-lamina by a DNA-bearing structure masked in situ in rat liver nuclei

We have developed a method by which nuclear shells containing nucleoli can be isolated from membrane-depleted rat liver nuclei. This method involves the removal of the internal chromatin. This chromatin is expelled from the nuclear shell using combinations of low and high ionic strength buffers. The expelled internal part is subsequently digested with DNase I or micrococcal nuclease. Examination by electron microscopy of the nuclear and the nucleolar structures at various steps of the isolation procedure shows that the nucleoli are anchored in the peripheral lamina by a pedicle that is continuous with an intranucleolar network. This network is masked in situ by nucleolar granules. The pedicle and the network which support the nucleolar DNA are composed mainly of non-histone proteins insoluble in 2M NaCl.     

Comparative studies on the electrophoretic patterns of acid-soluble chromosomal proteins duringZea mays early stages of embryo germination and root cell differentiation

Acid-soluble chromosomal proteins were extracted from purified nuclei, isolated from 3 – 4 h, 12 –14 h and 24 –26 h maize embryos, as well as from nuclei isolated from meris-tematic, elongating and differentiated cells, from 2 and 3-day-oldZea mays seedlings primary roots. Using polyacrylamide gel electrophoresis (urea-acetic acid in the cylindrical gels and slab-SDS-electrophoresis), it was established that germination and root cell differentiation induced changes in some nuclear acid-soluble chromosomal proteins, especially in histone H1. The results of proteins belonging to the high-mobility group proteins (HMG) and some other acid-soluble proteins with unknown nature for the plants, based on the electrophoretic mobility, were discussed.