The involvement of the root apex and cytokinins in the control of lateral root emergence in lettuce seedlings

Removal of the apical 3 mm of the primary root of hydroponically-grown lettuce seedlings 3 or 5 days after sowing, prevented further elongation of the root and increased both the number and total length of lateral roots. The length of the lateral zone, i.e. the distance from the base of the parent root to the lateral nearest the tip, except on one occasion, remained the same as the control in both 3 and 5 day treatments, until the length of the decapitated root (which had ceased elongating) became limiting.Zeatin applied via the roots, at a concentration range from 3 × 10^–10 M to 10^–8 M reduced tap root extension growth at all concentrations. Lateral root emergence was enhanced by low zeatin concentrations and retarded by higher ones. In general, the lateral zone length was the same in cytokinin-treated plants as in untreated controls.     

Changes in the Nucleus during Cellular Development in the Pea Seedling

Nuclei were isolated from three regions of the root and fromthe epicotyls of growing pea seedlings. As the dry mass of thenuclei was the same before and after isolation, and as the DNAcontent of the nuclei accounted for the DNA of the cells, theisolated nuclei are assumed to be a random sample of the nucleiin the intact tissues. The DNA, RNA, and protein contents percell were much greater in the epicotyl than in the root, butthe RNA and protein contents of the nucleus were similar inboth epicotyl and root. Although the amounts of DNA, RNA, andprotein per cell increased with increasing distance from theroot tip, increase in the protein content of the nucleus occurredmainly between the meristematic and expanding regions, and atthis same point nuclear RNA decreased. Nuclear volume increasedwith increasing distance from the root tip, whereas nucleardry mass remained virtually unchanged. The nucleus thereforebecomes increasingly hydrated as the cells mature. The datasuggest a changing interaction between the nucleus and the cytoplasmduring cellular development.     

Comparison of purified anti-actin and fluorescent-heavy meromyosin staining patterns in dividing cells

We purified actin antibodies by affinity chromatography from the serum of rabbits immunized with glutaraldehyde-fixed chicken gizzard actin filaments and used this anti-actin to localize actin in myofibrils and fixed cultured cells at each stage of the cell cycle. By double immunodiffusion the anti-actin reacted with both smooth and skeletal muscle actin. Several blocking and absorption experiments demonstrated that the antibodies also bound specifically to actin in nonmuscle cells. The same structures stained using either the direct or the indirect fluorescent antibody technique; and, while the indirect method was more sensitive, the direct method was superior because there was no detectable nonspecific staining. As expected, anti-actin stained the I-band of myofibrils. It also stained stress fibers and membrane ruffles in HeLa cells. Some PtK-2 cells have straight stress fibers which stained with anti-actin, but in confluent cultures all PtK-2 cells have, instead, sinuous phase-dense fibers which stained with antibody. At prophase the whole cytoplasm stained uniformly with anti-actin. During metaphase and anaphase, anti-actin staining was concentrated diffusely in the mitotic spindle. In contrast, fluorescent heavy meromyosin stained discrete fine spindle fibers in these fixed cells. During cytokinesis, anti-actin stained the whole cytoplasm uniformly and was not concentrated in the cleavage furrow.     

Hormonal factors controlling the initiation and development of lateral roots

As the first part of a comprehensive study of the hormonal control of lateral root initiation and development, the effect of surgical treatments such as removal of the root tip, one or more cotyledons, the young epicotyl, or combination of these treatments, on the induction and emergence of lateral roots on the primary root of pea seedlings has been examined. Results show that removal of the root tip leads to a rapid but transitory increase in the number of lateral primordia, the largest number arising in the most apical segment of decapitated roots suggesting the accumulation of acropetally moving promoter substances in this region. The cotyledons appear to be the main source of promoter substances for both the induction and emergence of lateral roots, although one or more promoters also appear to be produced in the epicotyl. The data further indicate that the root tip is possibly the source of a substance which moves basipetally and interacts with acropetally moving promoters to regulate the zone for lateral primordia initiation; the root tip also appears to be the source of a powerful inhibitor of lateral root emergence.     

Raffinose in seedlings of winter vetch (<Emphasis Type="Italic">Vicia villosa</Emphasis> Roth.) under osmotic stress and followed by recovery

During germination of winter vetch (Vicia villosa Roth.) seeds, the degradation of raffinose family oligosaccharides and galactosyl pinitols occurred faster in axis than in cotyledons. After 7 days of germination, all α-d-galactosides disappeared and the soluble carbohydrates in seedling tissues consisted of d-pinitol, sucrose, fructose, glucose and myo-inositol. Osmotic stress caused by incubation of seedlings in PEG 8000 solution (−0.5, −1.0, and −1.5 MPa) for 48 h induced the activity of crucial enzymes of the RFOs pathway, i.e. galactinol synthase and raffinose synthase, in both the root and epicotyl but not in cotyledons. The root and epicotyl accumulated elevated amounts of galactinol and raffinose as the osmotic potential was lowered. This process was transient because when PEG solution was replaced with water, galactinol and raffinose were degraded, thus confirming their direct involvement in the response of tissues to osmotic stress. Among other soluble carbohydrates, only sucrose accumulated in response to stress. The results did not show potential role of d-pinitol in the adjustment of winter vetch seedlings to osmotic stress.     

Effect of nickel on growth,proliferation, and differentiation of root cells in <Emphasis Type="Italic">Triticum aestivum</Emphasis> seedlings

The effect of 10^–6 and 10^–4 M NiSO₄ on cell growth, proliferation, and differentiation was studied over 48 h in seminal and lateral roots of five-day-old Triticum aestivum seedlings. 10^–6 M NiSO₄ did not significantly affect the root system, whereas 10^–4 M NiSO₄ inhibited its development. However, 10^–6 M NiSO₄ disturbed the contacts between the groups of closely related cells of the rhizodermis in the meristem. In the exodermis, an additional layer of cells was formed. At the nickel concentration of 10^–4 M, cell divisions in the outer layers of the root cells and metaxylem ceased earlier than in other root tissues positioned both centripetally and acropetally. Differentiation of protophloem sieve elements was completed in the meristem but at a greater distance from the root tip. Cell elongation started at the same distance from the root tip as in control plants. The rate of elongation decreased, and acropetally it stopped. Therefore, the cells of the xylem and metaphloem started to differentiate, and primordia of lateral roots were initiated and formed closer to the root tip. At a lethal concentration (10^–4 M), nickel induced necroses of elongating cells of the endodermis and pericycle. Nickel is supposed to enter the tissues of the central cylinder predominantly via the protoxylem and rapidly translocate along the xylem. As a result, the incubation of the roots at this concentration for 48 h almost did not affect the development of the phloem and probably sugar unloading, that makes possible to maintain the growth of meristematic cells and the cell division of the most important tissues for longer time.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 250–258.Original Russian Text Copyright © 2005 by N. Demchenko, Kalimova, K. Demchenko.This revised version was published online in April 2005 with a corrected cover date.     

Water potential, turgor and cell wall properties in elongating tissues of the hydrotropically bending roots of pea (Pisum sativum L.)

The hydrotropic bending of roots of an ageotropic pea mutant, ageotropum, was studied in humid air in a chamber with a steady humidity gradient. We examined the effects of atmospheric humidity around the root on the water status of root tissues, as well as the wall growth and the hydraulic properties of the elongating tissues. Atmospheric humidity at the surface of the root was clearly lower on the side orientated towards the air with lower humidity than on the side orientated towards the air with higher humidity. However, there were no differences in water potential and osmotic potential between the tissues that faced air with higher and lower humidities in the elongating and mature regions. Plastic extensibility was higher in the tissues that faced the air with lower humidity than in the tissues that faced the air with higher humidity. No differences in turgor pressure and yield threshold were observed between the tissues that faced air with higher and lower humidities. Therefore, the extensibility of the cell wall appeared to be responsible for the different growth rates of tissues in root hydrotropism. A further probable cause of the hydrotropical bending of roots is changes in the hydraulic conductance in the elongating tissues. Since the hydrotropic bending of roots occurred only when a root tip was exposed to a humidity gradient, hydrotropism might occur after perception of a difference in humidity by the root tip, with accompanying changes in cell wall extensibility and hydraulic conductance.     

Effect of root length on epicotyl dormancy release in seeds of Paeonia ludlowii,Tibetan peony

Background and Aims

Epicotyl dormancy break in seeds that have deep simple epicotyl morphophysiological dormancy (MPD) requires radicle emergence and even a certain root length in some species. However, the mechanisms by which root length affects epicotyl dormancy break are not clear at present. This study aims to explore the relationship between root length and epicotyl dormancy release in radicle-emerged seeds of Tibetan peony, Paeonia ludlowii, with discussion of the possible mechanisms.

Methods

Radicle-emerged seeds (radicle length 1·5, 3·0, 4·5 and 6·0 cm) were incubated at 5, 10 and 15 °C. During the stratification, some seeds were transferred to 15 °C and monitored for epicotyl–plumule growth. Hormone content was determined by ELISA, and the role of hormones in epicotyl dormancy release was tested by exogenous hormone and embryo culture.

Key Results

Cold stratification did not break the epicotyl dormancy until the root length was ≥6 cm. The indole-3-actic acid (IAA) and GA₃ contents of seeds having 6 cm roots were significantly higher than those of seeds with other root lengths, but the abscisic acid (ABA) content was lowest among radicle-emerged seeds. GA₃ (400 mg L⁻¹) could break epicotyl dormancy of all radicle-emerged seeds, while IAA (200 mg L⁻¹) had little or no effect. When grown on MS medium, radicles of naked embryos grew and cotyledons turned green, but epicotyls did not elongate. Naked embryos developed into seedlings on a mixed medium of MS + 100 mg L⁻¹ GA₃.

Conclusions

A root length of ≥6·0 cm is necessary for epicotyl dormancy release by cold stratification. The underlying reason for root length affecting epicotyl dormancy release is a difference in the GA₃/ABA ratio in the epicotyl within radicle-emerged seeds, which is mainly as a result of a difference in ABA accumulation before cold stratification.     

Distribution of polyamines and their related catabolic enzyme in etiolated and light-grown leguminosae seedlings

Diamine-oxidase (DAO; EC 1.4.3.6) activity and di-and polyamine levels were estimated along the epicotyl and root of light-grown and etiolated lentil (Lens culinaris Medicus) and pea (Pisum sativum L.) seedlings. The activity of DAO was higher in etiolated epicotyls than in lightgrown ones. In both species there was a positive correlation between DAO activity and the diamine (putrescine and cadaverine) levels along the whole epicotyl and root. Polyamine (spermine and spermidine) distribution seemed to be associated with the meristematic and elongating zone of the epicotyl and root. The physiological function of DAO is discussed in relation to its possible role in providing hydrogen peroxide to peroxidase-dependent reactions occurring in the cell wall.Abbreviations CAD cadaverine - DA diamine - DAO diamine oxidase - PA polyamine - PUT putrescine - SPD spermidine - SPM spermine     

The ancient subclasses of Arabidopsis Actin Depolymerizing Factor genes exhibit novel and differential expression

The Actin Depolymerizing Factor (ADF) gene family of Arabidopsis thaliana encodes 11 functional protein isovariants in four ancient subclasses. We report the characterization of the tissue-specific and developmental expression of all Arabidopsis ADF genes and the subcellular localization of several protein isovariants. The four subclasses exhibited distinct expression patterns as examined by qRT-PCR and histochemical assays of a GUS reporter gene under the control of individual ADF regulatory sequences. Subclass I ADFs were expressed strongly and constitutively in all vegetative and reproductive tissues except pollen. Subclass II ADFs were expressed specifically in mature pollen and pollen tubes or root epidermal trichoblast cells and root hairs, and these patterns evolved from an ancient dual expression pattern comprised of both polar tip growth cell types, still observed in the monocot Oryza sativa. Subclass III ADFs were expressed weakly in vegetative tissues, but were strongest in fast growing and/or differentiating cells including callus, emerging leaves, and meristem regions. The single subclass IV ADF was constitutively expressed at moderate levels in all tissues, including pollen. Immunocytochemical analysis with subclass-specific monoclonal antibodies demonstrated that subclass I isovariants localize to both the cytoplasm and the nucleus of leaf cells, while subclass II isovariants predominantly localize to the cytoplasm at the tip region of elongating root hairs and pollen tubes. The distinct expression patterns of the ADF subclasses support a model of ADF s co-evolving with the ancient and divergent actin isovariants.     

Nerve fibers in culture and their interactions with non-neural cells visualized by immunofluorescence

Cultures of embryonic mouse spinal cord explants, alone or in combination with rat myotubes, were stained by indirect immunofluorescence using antibodies against three structural proteins to: (a) reveal the distribution of these proteins among different cell types, and (b) test the usefulness of antibody staining to reveal the gross morphology of the neurite network in complex cultures. Affinity column purified antibodies were used against chicken gizzard actin, porcine brain tubulin, and skeletal muscle alpha-actinin. Neurites were stained intensely by anti-actin as was the stress fiber pattern of underlying fibroblasts. With anti-tubulin, the staining of neurites was an order of magnitude more intense than the staining of the microtubule pattern of background fibroblasts. Neurite cell bodies and astrocyte-like glia cells were stained with anti-tubulin and their nuclei remained unstained. Anti-tubulin could thus be used to trace even the finest extensions of nerve processes in spinal cord and spinal cord-muscle cultures. Furthermore, it could be combined with the histochemical reaction for acetylcholinesterase (AChE, EC 3.1.1.7) to demonstrate AChE-positive neurons and specialized nerve-muscle contact sites. The staining of neural elements with anti-alpha-actinin was generally much weaker than with anti-actin and anti-tubulin. Neurites were stained only moderately in comparison to myotube Z lines in the same culture. However, a distinct staining of the periphery of dorsal root ganglion cells was observed. Thus, a protein immunologically related to muscle alpha-actinin is present in the nervous system. In myotubes, Z lines were stained intensely with anti-alpha-actinin while I bands were only faintly stained with anti-actin. In isolated myofibrils, both structures were stained intensely with the same antibody preparations.     

Laser ablation ICP-MS reveals patterns of copper differing from zinc in growth zones of cucumber roots

Laser ablation coupled with inductively coupled plasma-mass spectrometry was used to find Cu and Zn concentration in surface tissue along a longitudinal developmental gradient with meristem, rapidly elongating tissue, and nongrowing tissue in a model system of seedling roots of Cucumis sativus L. (cucumber). Tissue metal accumulation was determined for roots of seedlings growing on cellulosic germination paper treated with nutrient solution (controls), and also treated with concentrations of Zn (40 ppm) and Cu (10 ppm) that reduced growth. Cu content of all roots is highest at the apex and falls sharply to lower values by 2 mm from the root tip. In contrast, at moderate Zn availability (0.07 ppm), Zn content rises from the apex to 2 mm then falls throughout the remainder of the growth zone. At high external Zn the spatial pattern resembles that of Cu. Cucumber root growth zones accumulate more of each metal with higher external availability. Metal deposition rates were calculated using a continuity equation with data on local metal content and growth velocity. Deposition rates of both metals are generally highest in the rapidly elongating region, 1.5–3.5 mm, even where metal concentration is decreasing with position and root age and even when the accumulation is inhibitory to growth.     

Anatomical observation of polyphenol changes in epidermal cells during the development of <Emphasis Type="Italic">Quercus acutissima</Emphasis>–<Emphasis Type="Italic">Scleroderma verrucosum</Emphasis> ectomycorrhizae

Quercus acutissima seedlings were cultivated in growth pouches and inoculated with Scleroderma verrucosum in order to assess the changes in polyphenol contents in epidermal cells during ECM development. Semithin sections stained with metachromatic Toluidine Blue O (TBO) were compared among non-inoculated lateral roots, early mantled lateral roots, and mycorrhizal roots with a mature mantle. Hyphae adhered closely or were embedded in mucilage-like materials on the epidermis. Epidermal cells and root hairs of the non-inoculated second-order lateral roots developing from the taproot harbored polyphenolic compounds that were stained by TBO. At non-inoculated stage, the average numbers of epidermal cells stained entirely (PC2), stained partially (PC1) or remaining unstained (PC0) were 16.5 ± 0.7, 0 ± 0, and 0 ± 0, respectively. At the early mantled stage, the numbers were 6.5 ± 1.6, 5.2 ± 1.4, and 4.2 ± 1.0, and at the mycorrhizal stage, it was 0 ± 0, 0 ± 0, and 32.8 ± 1.3 for PC2, PC1, and PC0, respectively. Total phenolic content in the root tips at each developmental stage declined with ECM development. The early mantled stage involved a dynamic process of polyphenol localization. However, some epidermal cells and endodermal cells of the proximal zone accumulated polyphenols. Eventually, polyphenolic compounds, which were found abundantly in the epidermal cells and root hairs of the non-inoculated lateral roots of the host, disappeared at the mycorrhization process with the symbiont.     

Immunolabeled microtubules and microfilaments are visible in multiple cell layers of rye root tip sections

Summary A protocol was developed to observe plant microtubules and actin microfilaments in large tissue samples without physical sectioning. Rye (Secale cereale L. cv. Rymin) root tip pieces from two-day-old seedlings were fixed and processed for immunolabeling. Incubation times of 24–48 h were required to insure adequate penetration of fixatives, antibodies, and washing buffers. Clearing of the tissue with methyl salicylate reduced background auto-fluorescence that would otherwise interfere with the resolution of cytoskeletal structures. Microtubules or microfilaments in 5–7 cell layers were visualized using the optical-sectioning capability of laser scanning confocal microscopy (LSCM) and projected as three-dimensional images. The three-dimensional character of the cytoskeletal elements is retained when viewing stained cells of intact tissue. The net-like character of a microfilament array radiating out from a single point into the cytoplasm is maintained when the cells are stained in intact root tip pieces and imaging is accomplished in situ.Abbreviations Cy3 cyanine 3.18-conjugated goat anti-mouse IgG - FITC-M fluorescein isothiocyanate conjugated anti-mouse IgG - IFB immunofluorescence buffer - LSCM laser scanning confoeal microscopy - TPBS phosphate-buffered saline with 0.1% Triton X-100     

Cadmium can induce alterations in the cellular localization and expression of three major nucleolar proteins in root tip cells of Vicia faba L

Aims

The findings of our earlier investigations indicated that cadmium (Cd) could induce some nucleolar materials containing the argyrophilic proteins scattered in the nuclei and located in cytoplasm in the root tip cells of Allium sative and Vicia faba. However, what kinds of nucleolar proteins are affected has not been reported up to now. In order to further confirm the cytological effects of Cd on nucleolus and nucleolar proteins, alterations in the cellular localization and expression of three major nucleolar proteins: nucleophosmin, fibrillarin and nucleolin were investigated under the treatment with Cd in the root tip cells of V. faba in this study.

Methods

Silver methods, Indirect immunofluorescent microscopy; Western blotting.

Results

The obvious toxic effects on nucleoli in the cells were observed after Cd treatment. Nucleolar proteins, nucleophosmin, fibrillarin, and nucleolin in root tip cells exposed to Cd (50 μM) for 48 h were translocated from nucleolus to nucleoplasm and cytoplasm and expression of the three major nucleolar proteins was relatively higher when compared with control.     

Cell division in Lemna minor roots treated with lead

Treatment of Lemna minor L. roots with 15 μM Pb²⁺ supplied as Pb(NO₃)₂ in 50-fold diluted Wang medium caused a progressive reduction in mitotic activity in the root tip. The percentage of dividing nuclei after 1, 6, 12 and 12 h of lead treatment was 6.25, 4.4, 3.4 and 0.3, respectively as compared to 7.1–7.7% in the control. After 6 h of lead treatment the number of cells in metaphase and anaphase was reduced by four- and nine-fold, respectively and after 12 h these phases were not detected. There were 3- and 10-fold fewer cells in telophase after 6 and 24 h, while those in prophase were reduced only in the 24 h treatment (a 30-fold reduction). These effects were associated with an increase in the number of cells exhibiting disturbances including lagging chromosomes, chromosome bridges, micronuclei, and nuclei with more condensed chromatin. The formation of micronuclei in root cells of L. minor cells at a very low dose of lead indicates that roots of this aquatic plant may be more sensitive to lead than those of terrestrial plants.     

Effects of Copper on Root Growth, Cell Division, and Nucleolus of Zea Mays

The effects of different concentrations (10^–5 – 10^–2 M) of copper sulfate on root growth, cell division and nucleoli in root tip cells of Zea mays L. were investigated. 10^–5 M Cu stimulated root growth, but at higher concentrations (10^–4 – 10^–2 M) inhibited it. Cu had toxic effects on chromosomal morphology: c-mitosis, anaphase bridges, and chromosome stickiness were induced. Some nuclei had irregular shape and particles extruded from nucleoli to nuclei and finally from the nuclei into the cytoplasm.