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PubMed1997

Gravitropism in tip-growing cells.

Unicellular tip-growing cells are excellent experimental systems in which to study gravitropism because cell extension, gravity sensing and the gravity response are all confined to the apical dome. Thus various approaches can be used to determine the distinct steps of the short gravitropic signal-transduction chain, which lacks a signal-transmission phase between the gravity-sensing cells and the competent responding target cells. Single-cell systems readily allow in-vivo observation of cellular processes during gravistimulation at 1 g, centrifugation, clinostatting and in microgravity, as well as permitting fluorescence labeling. Such diverse studies have revealed fascinating information on the mechanism of gravitropic tip growth, especially on the important role of the cytoskeleton in the positioning of the statoliths and in organizing and adjusting the Spitzenkorper. A hypothesis explaining the negative and positive gravitropism of Chara rhizoids and Chara protonemata has been put forward, which emphasizes the role of the actin cytoskeleton in the process of gravitropic tip-growth. Differences in the gravitropic responses of single-cell systems, however, reflect a diversity of gravitropic mechanisms, and represent an example of parallel evolution.

Bryopsida↗
PubMed1997

Calcium in gravitropism. A re-examination.

For more than a decade it has been assumed that there is a strong relationship between Ca2+ and gravitropism. There is evidence to suggest that the movement of Ca2+ in the wall might regulate extension growth and that free intracellular Ca2+ might mediate signalling in statocytes. However, it is unlikely that changes in either wall Ca2+ or the concentration of free intracellular calcium, [Ca2+]i, act independently of each other. This results in ambiguity in both experimentation and interpretation of experimental information. It is also uncertain that the observed redistributions of wall Ca2+ are sufficient to induce bending and there is at present no direct evidence from measurement or imaging that changes in [Ca2+]i initiate or transduce gravitropic signals. The evidence relating Ca2+ to gravitropism is therefore substantial but still circumstantial. The involvement of free intracellular Ca2+ might best be tested using the new technologies of transgenic aequorin targeted to columella cells and thus resolve this important question. New information which relates control or orientation of tip-growing cells, such as pollen tubes, to cytosolic Ca2+ manipulation is presented. This information may be directly applicable to a gravitropic model system studied for many years, the Chara rhizoid.

Calcimycin↗
PubMed2003

Chilling stability of microtubules in root-tip cells of cucumber.

The chilling stability of microtubules was investigated in root-tip cells of cucumber (Cucumis sativus L.) using immunofluorescence labeling and a confocal laser scanning microscope. After low temperature (4 degrees C) treatment for 5 h, we found not only chilling-stable cortical microtubules under the plasma membrane of the root-tip cells, but also chilling-stable punctate microtubules in the cytoplasm, which might be the microtubules associated with organelles. 6-Dimethylaminopurine (6-DMAP), an inhibitor of protein kinase, reduced the chilling stability of the cortical microtubules in the cells. When the root-tip cells were restored to normal growth temperature (28 degrees C), the recovery of the microtubules seemed to be closely associated with the microtubules still remaining in these cells after chilling treatment.

Adenine↗
PubMed2001

Endoplasmic reticulum preprophase band in dividing root-tip cells of Pinus brutia.

In dividing root-tip cells of Pinus brutia Ten., immunolocalization of the luminal endoplasmic reticulum (ER) proteins, which have the C-terminal HDEL sequence, reveals that the ER is reorganized during the preprophase/prophase stage. Portions of ER were arrayed into a ring-like structure at the site of the microtubule preprophase band (Mt-PPB). This preprophase ER band (ER-PPB) resembles that of the Mt-PPB. The former undergoes a maturation process closely similar to that of the latter. Our data show that the PPB region has a more complex organization than is currently believed. The probable function(s) of the ER-PPB is discussed.

Actin Cytoskeleton↗
PubMed2003

Preparation of chromosomes from plant leaf meristems for karyotype analysis and in situ hybridization.

A reliable method for preparing metaphase chromosomes from plant leaf tissues is described. The chromosomes are suitable for karyotype analysis and gene mapping by fluorescence in situ hybridisation (FISH). The method is based on enzymatic digestion of young leaf tissues (shoot-tips) after which the resulting protoplasts are treated hypotonically before being dropped onto microscopic slides. Compared to root-tip chromosomes, leaf chromosomes tend to be longer, or less condensed, and hence more karyotypically differentiated. Metaphase index in young leaf tissues is also very high. Metaphase spread consists of evenly and well-distributed chromosomes and this allows accurate counting. The plant used to demonstrate this method is birch (Betula L.), a group of tree species that has extremely small chromosomes. Root-tip chromosomes of these plants are difficult to obtain, as cutting does not produce roots readily. Seedling chromosomes do not represent the same genomic constitution as their mother trees due to introgressive hybridisation. Furthermore, sample collection in the field is convenient and actively growing leaf buds are available throughout the growing season. FISH experiments with these leaf chromosomes also give good results comparable to those obtained with root-tip chromosomes or even better as mapping on long or extended chromosomes has high resolution in general. Mapping of the 16S-28S ribosomal genes on birch leaf chromosomes has been shown to differentiate between birch species and therefore can accurately confirm their interspecific hybrids.

Betula↗
PubMed1990

Actin filaments responsible for the location of the nucleus in the lentil statocyte are sensitive to gravity.

The location of the nucleus in statocytes or lentil roots grown: 1), at 1 g on the ground, 2), on a 1 g centrifuge in space, 3), in simulated microgravity on a slowly rotating clinostat (0.9 rmp) 4), in microgravity in space was investigated and statistically evaluated. In cells differentiated at 1 g on the ground, the nuclear membrane was almost in contact with the plasmalemma lining the proximal cell wall, whereas in statocytes of roots crown on the clinostat there was a distance of 0.47 micrometers (horizontal clinorotation) and or 0.76 micrometers (vertical clinorotation) between these membranes. However, in microgravity the nucleus was the most displaced, 0.87 micrometers from the proximal cell wall. Centrifugation of vertically grown roots in the root-tip direction showed that the threshold of centrifugal force to detach all nuclei from the proximal cell wall was about 40 g. In statocytes developed in the presence of cytochalasin B at 1 g the nuclei were sedimented on the amyloplasts at the distal cell pole, demonstrating that the location of the nucleus depends on actin filaments. The results obtained are in agreement with the hypothesis that gravity causes a tension of actin filaments and that this part of the cytoskeleton undergoes a relaxation in microgravity.

Actins↗
PubMed1984

The role of calcium ions in cytological effects of hypogravity.

Electron-cytochemical and biochemical methods made it possible to reveal certain differences in ATPase activity stimulation by calcium ions in root apex cells of pea seedlings and moss protonema Funaria hygrometrica grown under stationary and slow clinostatic (2 rev/min) conditions. It was showed that under clinostatic conditions in comparison with the control variant the ATPase activity decreases in plasmalemma. The protein content in the plasmalemma fraction was also twice as low under these conditions. The root apex cells of the pea seedlings grown under spaceflight conditions were found to contain high concentrations of membrane-bound calcium. The data obtained are discussed in relation to problems of possible mechanisms of disturbance in calcium balance and the system of active calcium ion transport through plasmalemma under hypogravity.

Bryopsida↗
PubMed2005

Comparative biomonitoring of leachates from hazardous solid waste of two industries using Allium test.

Hazardous industrial wastes are inevitable source of environmental pollution. Leachates from these wastes might contaminate the origins of potable water and affect human health. The study was carried out to determine the possible genotoxic effects of leachates from solid waste of a metal and dye industry using the Allium cepa chromosome aberrations assay. The 10% leachates were prepared from solid wastes obtained from both the industries and examined for the presence of heavy metal content and genotoxicity. To simulate the field and laboratory conditions, A. cepa bulbs were exposed through soil and aqueous medium for 48 h to 2.5-10% leachates. The results revealed that both metal waste leachate (MWL) and dye waste leachate (DWL) contained high concentrations of chromium, nickel and iron that significantly induced cytogenetic alterations. Significant inhibition of mitotic index (MI), inductions of chromosomal/mitotic aberrations (CA/MA) and micronuclei (MN) formation were found in all experimental groups exposed to MWL and DWL. The effects observed were concentration dependent and the frequency of aberrations was higher with treatment of MWL than DWL. The MI was severely inhibited at 10% aqueous exposure it was 4.59+/-0.69 (P<0.001) in MWL and almost half to that induced by DWL that was 8.62+/-0.69 (P<0.05). Significant frequency of CA/MA and MN induced by MWL was 14.21 (P<0.001) and 0.33 (P<0.001) whereas CA/MA and MN induced by DWL was 7.81 (P<0.001) and 0.13 (P<0.05) in the aqueous medium. The investigations inferred that abnormalities caused by MWL were higher than DWL both in soil and aqueous media. These toxic responses may have relied on raised heavy metal concentrations of metal-based than dye industrial wastes.

Chromosome Aberrations↗
PubMed2001

Gravitropism of axial organs in multicellular plants.

Gravitropism of plant organs such as roots, stems and coleoptiles can be separated into four distinct phases: 1. perception (gravity sensing), 2. transduction of a signal into the target region and 3. the response (differential growth). This last reaction is followed by a straightening of the curved organ (4.). The perception of the gravitropic stimulus upon horizontal positioning of the organ (1.) occurs via amyloplasts that sediment within the statocytes. This conclusion is supported by our finding that submerged rice coleoptiles that lack sedimentable amyloplasts show no graviresponse. The mode of signal transduction (2.) from the statocytes to the peripheral cell layers is still unknown. Differential growth (3.) consists of a cessation of cell expansion on the upper side and an enhancement of elongation on the lower side of the organ. Based on the facts that the sturdy outer epidermal wall (OEW) constitutes the growth-controlling structure of the coleoptile and that growth-related osmiophilic particles accumulate on the upper OEW, it is concluded that the differential incorporation of wall material (presumably glycoproteins) is causally involved. During gravitropic bending, electron-dense particles ('wall-loosening capacity') accumulate on the growth-inhibited upper OEW. It is proposed that the autotropic straightening response, which is in part due to an acceleration of cell elongation on the curved upper side, may be attributable to an incorporation of the accumulated particles ('release of wall-loosening capacity'). This novel mechanism of autotropic re-bending and its implications for the Cholodny-Went hypothesis are discussed.

Cotyledon↗
PubMed1997

Application of SSNTDs in radiobiological investigations aboard recoverable satellites.

In recent years some Biostack experiments including a wide spectrum of biological objects have been devoted to study of the radiobiological effects on dry seeds aboard recoverable satellites. Some impressive phenomena have been observed. Clearly, the large amount of energy deposited by the highly ionizing heavy nuclei of cosmic rays is the principal reason for the induced aberrations of the chromosomes of wheat root tip cells. A methodical description of the experimental arrangement and procedure of handling and evaluation of given. The preliminary physical and biological results from the experimental "wheat seeds" are presented.

Chromosome Aberrations↗
PubMed2000

The role of root border cells in plant defense.

The survival of a plant depends upon the capacity of root tips to sense and move towards water and other nutrients in the soil. Perhaps because of the root tip's vital role in plant health, it is ensheathed by large populations of detached somatic cells - root 'border' cells - which have the ability to engineer the chemical and physical properties of the external environment. Of particular significance, is the production by border cells of specific chemicals that can dramatically alter the behavior of populations of soilborne microflora. Molecular approaches are being used to identify and manipulate the expression of plant genes that control the production and the specialized properties of border cells in transgenic plants. Such plants can be used to test the hypothesis that these unusual cells act as a phalanx of biological 'goalies', which neutralize dangers to newly generated root tissue as the root tip makes its way through soil.

Plant Root Cap↗
PubMed1999

Cell cycle synchronization in plant root meristems.

The analysis of structure and metabolism of a cell at a defined phase of cell cycle is often difficult because cell cycle progression in somatic tissues is asynchronous and only a fraction of cells are cycling. An elegant solution to obtain populations of cells enriched for single stage of the cell cycle is to impose the synchrony artificially. Different systems have been used to obtain synchronized populations of plant cells, including suspension-cultured cells, leaf mesophyll protoplasts and root tip meristems. Root tips have been frequently used in a variety of studies ranging from chromosome analysis to cell cycle and its regulation. Seedlings with actively growing roots may be obtained in most plant species, they are easy to handle, the experimental system is well defined, reproducible and can be easily modified for different species. This paper describes a protocol for cell cycle synchronization in root tips of Vicia faba, which is based on the use of DNA synthesis inhibitor hydroxyurea [18]. Modifications of the protocol for Pisum sativum, Medicago sativa, Hordeum vulgare, Secale cereale, Triticum aestivum, and Zea mays are also given. Flow cytometric data indicate that about 90% of root tip cells are synchronized. On average, mitotic indices exceeding 50% are obtained with the method. Synchronized cells may be accumulated at metaphase using a mitotic spindle inhibitor to achieve metaphase indices exceeding 50%.

Genes, cdc↗
PubMed1999

Bivariate flow cytometry DNA/BrdUrd analysis of plant cell cycle.

We describe a protocol for flow cytometry analysis of cell cycle in plants using indirect immunolabelling staining and Vicia faba, Pisum sativum and Zea mays root tip cells as model systems. The protocol is based on simultaneous analysis of two fluorescent signals. The first, obtained after staining with propidium iodide, is used to quantify nuclear DNA content. The second, obtained after indirect immunofluorescent staining of bromodeoxyuridine (BrdUrd), is used to quantify the amount of BrdUrd incorporated into nuclear DNA. In an attempt to standardize the procedure, the effects of various conditions for partial DNA denaturation using HCl, as well as of BrdUrd concentration and incorporation time on flow cytometry DNA/BrdUrd content analysis have been studied. Maximum BrdUrd-linked fluorescence was observed after a 30 min pulse with 10 microM BrdUrd and after DNA denaturation with 1.5 N HCl (final concentration) for 30 min at 25 degrees C. Under these conditions, DNA content histograms with relatively small coefficient of variation (< 4%, full peak) could be obtained. To avoid non-specific staining of cytoplasm and cell walls, the protocol involves the use of nuclei isolated from formaldehyde-fixed tissues. Fixed isolated nuclei are stable and may be stored in hexylene glycol 0.75 M at 4 degrees C for prolonged periods prior to actual staining and analysis.

Bromodeoxyuridine↗
PubMed1999

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.

Antibodies↗
PubMed2001

Changes in growth and structure of pea primary roots (Pisum sativum L. cv. Alaska) as a result of sudden flooding.

Pea (Pisum sativum L. cv. Alaska) primary roots were exposed to flooding after growth for 4 or 5 d at 25 degrees C under relatively dry conditions. Flooding after 4 d growth reduced, but did not stop, primary root growth, and cavities caused by degradation of central vascular cells were typically found from 10-60 mm from the tips. Flooding after 5 d stopped primary root growth and caused cell death in the tips, and vascular cavities formed that typically were 20-60 mm from the tips of the roots. Degradation of root tip cells in 5-day-roots was very rapid and began in the elongation zone and later in the apical zone. Root tips discolored, narrowed or curled before growth arrest. The mitotic indices of 5-day-root tips were suppressed by the flooding treatment. A few mitotic figures were observed in roots treated with flooding after 4 d growth. Affected cells had condensed nuclei, but cytoplasms appeared to be normal in the early stages of cell degradation. Later these cells became very vacuolated. The relationship of flooding to root growth, vascular cavity formation, and the morphology of pea primary roots is described with regard to the ability to resist flooding stress.

Apoptosis↗
PubMed2001

Mastoparan alters subcellular distribution of profilin and remodels F-actin cytoskeleton in cells of maize root apices.

Indirect immunofluorescence localization of profilin in cells of maize root apices revealed that this abundant protein was present both in the cytoplasm and within nuclei. Nucleo-cytoplasmic partitioning of profilin exhibits tissue-specific and developmental features. Mastoparan-mediated activation of heterotrimeric G-proteins, presumably through triggering a phosphoinositide-signaling pathway based on phosphatidylinositol-4,5-bisphosphate (PIP(2)), induced relocalization of profilin from nuclei into the cytoplasm of root apex cells. In contrast, PIP(2) accumulated within nuclei of mastoparan-treated root cells. Intriguingly, cytoplasmic accumulation of profilin was associated with remodeling of F-actin arrays in root apex cells. Specifically, dense F-actin networks were dismantled and distinct actin patches became associated with the periphery of small vacuoles. On the other hand, disruption of F-actin with the G-actin sequestering agent latrunculin B does not affect the subcellular distribution of profilin or PIP(2). These data suggest that nuclear profilin can mediate a stimulus-response action on the actin cytoskeleton which is somehow linked to a phosphoinositide-signaling cascade.

Actins↗
PubMed2002

Hyperosmotic stress induces formation of tubulin macrotubules in root-tip cells of Triticum turgidum: their probable involvement in protoplast volume control.

Treatment of root-tip cells of Triticum turgidum with 1 M mannitol solution for 30 min induces microtubule (Mt) disintegration in the plasmolyzed protoplasts. Interphase plasmolyzed cells possess many cortical, perinuclear and endoplasmic macrotubules, 35 nm in mean diameter, forming prominent arrays. In dividing cells macrotubules assemble into aberrant mitotic and cytokinetic apparatuses resulting in the disturbance of cell division. Putative tubulin paracrystals were occasionally observed in plasmolyzed cells. The quantity of polymeric tubulin in plasmolyzed cells exceeds that in control cells. Root-tip cells exposed for 2-8 h to plasmolyticum recover partially, although the volume of the plasmolyzed protoplast does not change detectably. Among other events, the macrotubules are replaced by Mts, chromatin assumes its typical appearance and the cells undergo typical cell divisions. Additionally, polysaccharidic material is found in the periplasmic space. Oryzalin and colchicine treatment induced macrotubule disintegration and a significant reduction of protoplast volume in every plasmolyzed cell type examined, whereas cytochalasin B had only minor effects restricted to differentiated cells. These results suggest that Mt destruction by hyperosmotic stress, and their replacement by tubulin macrotubules and putative tubulin paracrystals is a common feature among angiosperms and that macrotubules are involved in the mechanism of protoplast volume regulation.

Adaptation, Physiological↗
PubMed2001

Root mucilage from pea and its utilization by rhizosphere bacteria as a sole carbon source.

Plant roots secrete a complex polysaccharide mucilage that may provide a significant source of carbon for microbes that colonize the rhizosphere. High molecular weight mucilage was separated by high-pressure liquid chromatography gel filtration from low molecular weight components of pea root exudate. Purified pea root mucilage generally was similar in sugar and glycosidic linkage composition to mucilage from cowpea, wheat, rice, and maize, but appeared to contain an unusually high amount of material that was similar to arabinogalactan protein. Purified pea mucilage was used as the sole carbon source for growth of several pea rhizosphere bacteria, including Rhizobium leguminosarum 8401 and 4292, Burkholderia cepacia AMMD, and Pseudomonas fluorescens PRA25. These species grew on mucilage to cell densities of three- to 25-fold higher than controls with no added carbon source, with cell densities of 1 to 15% of those obtained on an equal weight of glucose. Micromolar concentrations of nod gene-inducing flavonoids specifically stimulated mucilage-dependent growth of R. leguminosarum 8401 to levels almost equaling the glucose controls. R. leguminosarum 8401 was able to hydrolyze p-nitrophenyl glycosides of various sugars and partially utilize a number of purified plant polysaccharides as sole carbon sources, indicating that R. leguminosarum 8401 can make an unexpected variety of carbohydrases, in accordance with its ability to extensively utilize pea root mucilage.

Amino Acids↗