Apical meristem organization and lack of establishment of the quiescent center in Cactaceae roots with determinate growth

Some species of Cactaceae from the Sonoran Desert are characterized by a determinate growth pattern of the primary root, which is important for rapid lateral-root formation and seedling establishment. An analysis of the determinate root growth can be helpful for understanding the mechanism of meristem maintenance in plants in general. Stenocereus gummosus (Engelm.) Gibson & Horak and Pachycereus pringlei (S. Watson) Britton & Rose are characterized by an open type of root apical meristem. Immunohistochemical analysis of 5-bromo-2-deoxyuridine incorporation into S. gummosus showed that the percentage of cells passing through the S-phase in a 24-h period is the same within the zone where a population of relatively slowly proliferating cells could be established and above this zone in the meristem. This indicated the absence of the quiescent center (QC) in S. gummosus. During the second and the third days of growth, in the distal meristem portion of P. pringlei roots, a compact group of cells that had a cell cycle longer than in the proximal meristem was found, indicating the presence of the QC. However, later in development, the QC could not be detected in this species. These data suggest that during post-germination the absence of the establishment of the QC within the apical meristem and limited proliferative activity of initial cells are the main components of a determinate developmental program and that establishment of the QC is required for maintenance of the meristem and indeterminate root growth in plants.Abbreviations QC quiescent center - RCP root cap-protoderm - BrdU 5-bromo-2-deoxyuridine - FITC fluorescein isothiocyanate - DAPI 4,6-diamidino-2-phenylindole     

Origin of the quiescent center in the root of Capsella bursa-pastoris (L.) Medik

The origin of the quiescent center in the embryonic radicle of Capsella bursa-pastoris was investigated by in-situ hybridization to cellular polyadenylic-acid-containing RNA using [³H]polyuridylic acid as a probe. In the globular embryo, autoradiographic silver grains were localized in all cells of the presumptive root apex except in the hypophysis. As the inner cell formed by a transverse division of the hypophysis cut off new cells toward the central procambial cylinder of the embryo, these cells remained characteristically unlabeled, in contrast to the labeled cells of the rest of the embryo. In the embryonic radicles of mature seeds and of seedlings, cells derived from the hypophysis appeared as a nonmeristematic, unlabeled, hemispherical group, bounded by the procambium to the inside and the root epidermis to the outside. When root tips excised from 2-d-old seedlings were incubated in [methyl-³H]thymidine, sectioned, and autoradiographed, cells derived from the inner cell of the hypophysis were found to be unlabeled, thus showing that they constitute the specific cells of the quiescent center. These results present evidence for the single-cell origin of the quiescent center in an angiosperm root and a role for the hypophysis in it.Abbreviations poly(A)⁺RNA polyadenylicacid-containing RNA - [³H]poly(U) [³H]polyuridylic acid - QC quiescent center This work was supported in part by National Science Foundation grants PCM-7902898 and DCB-8709092.     

Inhibition of maize root growth by high nitrate supply is correlated with reduced IAA levels in roots

The plant root system is highly sensitive to nutrient availability and distribution in the soil. For instance, root elongation is inhibited when grown in high nitrate concentrations. To decipher the mechanism underlying the nitrate-induced inhibition of root elongation, the involvement of the plant hormone auxin in nitrate-dependent root elongation of maize was investigated. Root growth, nitrogen and nitrate concentrations, and indole-3-acetic acid (IAA) concentrations in roots and in phloem exudates of maize grown under varying nitrate concentrations were analyzed. Total N and nitrate concentrations in shoots and roots increased and elongation of primary, seminal and crown roots were inhibited with increasing external nitrate from 0.05 to 5 mM. High nitrate-inhibited root growth resulted primarily from the reduced cell elongation and not from changes in meristem length. IAA concentrations in phloem exudates reduced with higher nitrate supply. Inhibition of root growth by high nitrate was closely related to the reduction of IAA levels in roots, especially in the sections close to root tips. Exogenous NAA and IAA restored primary root growth in high nitrate concentrations. It is concluded that the inhibitory effect of high nitrate concentrations on root growth may be partly attributed to the decrease in auxin concentrations of roots.     

Three maize root-specific genes are not correctly expressed in regenerated caps in the absence of the quiescent center

The quiescent center is viewed as an architectural template in the root apical meristem of all angiosperm and gymnosperm root tips. In roots of Arabidopsis thaliana (L.) Heynh., the quiescent center inhibits differentiation of contacting initial cells and maintains the surrounding initial cells as stem cells. Here, the role of the quiescent center in the development of the maize (Zea mays L.) root cap has been further explored. Three maize root-specific genes were identified. Two of these were exclusively expressed in the root cap and one of them encoded a GDP-mannose-4,6-dehydratase. Most likely these two genes are structural, tissue-specific markers of the cap. The third gene, a putative glycine-rich cell wall protein, was expressed in the cap and in the root epidermis and, conceivably is a positional marker of the cap. Microsurgical and molecular data indicate that the quiescent center and cap initials may regulate the positional and structural expression of these genes in the cap and thereby control root cap development. Received: 22 September 1999 / Accepted: 9 November 1999     

A negative image of the quiescent centre in regenerating root apices of Zea mays

Usually the presence of the quiescent centre in roots is demonstrated by the absence of labelled nuclei following treatment of the root with appropriate radioactive markers. By modification of the pulselabelling technique, a negative image of the quiescent center, showing more intense labelling from [³H]thymidine than the surrounding area, was obtained in regenerating root apices of Zea mays L.     

Hydroxyurea-induced mitotic synchronization in Allium sativum root meristems

The synchronized divisions following a treatment with hydroxyurea (HU) — an inhibitor of DNA synthesis — were studied in root meristems of Allium sativum using two methods: autoradiography of median sections and morphological labeling with a cytokinesis inhibitor. It is shown that the second wave of mitoses is heterogeneous: it is composed mostly of cells which have been synchronized in the S phase by the HU treatment, of cells coming from the quiescent center stimulated to enter DNA synthesis and of cells which were not blocked by the 23 h HU treatment (slow cycling cells). It is also shown that the cell cycle following the first synchronized division is considerably shortened by the synchronization procedure.Abbreviations QC quiescent center - HU hydroxyurea - MHQD methyl-3 hydroxy-6 quinazoline dione 2–4     

Effect of mitotic inhibitors on the ultrastructure of root meristem cells

After 5 h in 10^-3M vinblastine the most obvious effects upon Vicia faba L. cells are seen in the spindle apparatus. These include the microtubules themselves as indicated by c-type metaphases and the pole regions of otherwise intact spindles, leading to multipolar anaphases and to telophases with more than two daughter nuclei. Vesicle transport may be undisturbed and new cell walls can be formed, although cases of disturbed cell plate and cell wall formation were noted occasionally, accompanied by myelinizations in phragmosomes. After 24 h in the same concentration of vinblastine, divisions are no longer observed and the plasma membranes are severely affected. They show extensive myelinizations, accumulations of lipids and dehiscence from the cell walls which are frequently thickened and irregularly formed. Of the other cellular membranes, the nuclear envelope and, more frequently, the tonoplast may be affected. Electron-dense deposits appear in the vacuoles. Comparable, though less severe, changes including multipolar anaphases and myelinizations result from treatment with lumicolchicine, but not with colchicine. Vinblastine leads to the appearance of filament bundles both in cytoplasm and karyoplasm, lumicolchicine to morphologically identical filaments in the cytoplasm alone. The nature of these filaments is unknown.Abbreviation VLB vincaleukoblastine     

The role of the epidermis and cortex in gravitropic curvature of maize roots

In order to determine the role of the epidermis and cortex in gravitropic curvature of seedling roots of maize (Zea mays L. cv. Merit), the cortex on the two opposite flanks was removed from the meristem through the growing zone; gravitropic curvature was measured with the roots oriented horizontally with the cut flanks either on the upper and lower side, or on the lateral sides as a wound control. Curvature was slower in both these treatments (53° in 5 h) than in intact roots (82°), but there was no difference between the two orientations in extent and rate of curvature, nor in the latent time, showing that epidermis and cortex were not the site of action of the growth-regulating signal. The amount of cortex removed made no difference in the extent of curvature. Curvature was eliminated when the endodermis was damaged, raising the possibility that the endodermis or the stele-cortex interface controls gravitropic curvature in roots. The elongation rate of roots from which just the epidermis had been peeled was reduced by 0.01 mM auxin (indole-3-acetic acid) from 0.42 to 0.27 mm h^-1, contradicting the hypothesis that only the epidermis responds to changes in auxin activity during gravistimulation. These observations indicate that gravitropic curvature in maize roots is not driven by differential cortical cell enlargement, and that movement of growth regulator(s) from the tip to the elongating zone is unlikely to occur in the cortex.Abbreviations df degrees of freedom - IAA indole-3-acetic acid     

Light-regulated protein and mRNA synthesis in root caps of maize

Illumination of maize roots initiates changes in mRNA levels and in the activities of proteins within the root cap. Using Northern analysis we showed a 5–6-fold increase in the levels of three specific mRNAs and a 14-fold increase in plastid mRNA. This increase is rapid, occurring within 30 minutes of illumination. With prolonged periods of darkness following illumination, messages return to levels observed in dark, control caps. For two species of mRNA illumination results in a reduction in message levels. Light-stimulated increases in the levels of specific mRNAs are proportionally greater than are increases in the activities of corresponding proteins. We suggest that the light-stimulated increase in protein activity in root caps may be preceded by and occur as a consequence of enhanced levels of mRNA. Our work suggests that photomorphogenesis in roots could involve changes in the levels of a wide variety of mRNAs within the root cap.     

Importance of the cap in maize root growth

A large population of primary roots of Zea mays (cv. LG 11) was selected for uniform length at zero time. Their individual growth rates were measured over an 8-h period in the vertical position (in humid air, darkness). Three groups of these roots with significantly different growth rates were then chosen and their cap length was measured. It was found that slowly growing roots had long caps whereas rapidly growing roots had short caps. The production by the cap cells of basipetally transported growth inhibitors was tested (biologically by the curvature of half-decapped roots) and found to be significantly higher for longer root caps than that for shorter ones.     

Auxin binding in roots: A comparison between maize roots and coleoptiles

Auxin binding onto membrane fractions of primary roots of maize seedlings has been demonstrated using naphth-1yl-acetic acid (NAA) and indol-3yl-acetic acid (IAA) as ligands. This binding is compared with the already well characterized interaction between auxins and coleoptile membranes. The results indicate that while kinetic parameters are of the same order for root and coleoptile binding, a number of differences occur with respect to location in cells and relative affinity. The possible significance of the existence of such binding sites in root cells is discussed in relation to auxin action.Abbreviations 4-Cl-PA 4-chlorophenoxyacetic acid - EDTA ethylene diamine tetracetic acid - IAA indol-3yl-acetic acid - MCPA 2-methyl-4-chlorophenoxyacetic acid - NAA naphth-1yl-acetic acid - 2-NAA naphth-2yl-acetic acid - Tris 2-amino-2-(hydroxymethyl) propane-1,3 diol - TIBA 2,3,5 triiodobenzoic acid - NPA naphthylphthalamic acid - PCIB 4-chlorophenoxyisobutyric acid - PCPP 4-chlorophenoxyisopropionic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Comparison of maize root mucilages isolated from root exudates and root surface extracts by complementary cytological and biochemical investigations

Summary A strategy to obtain fractions enriched in mucilages secreted by root caps or produced by the rhizodermis of axenicallygrown maize seedlings is proposed. It involves a two-step procedure allowing the successive collection of root exudates and surface extracts from the same set of intact, sterile maize plants. Cytological controls were performed at each phase of collection. Whereas root cap mucilage is easily collected in water after one day's extraction, under conditions favouring secretory activity, rhizodermal mucilage remains tightly adherent to the root surface. It can be better extracted using neutral saline buffer assisted by gentle shaking at low temperature. Acidic saline buffer is unsuitable as it induces cell lysis and release of cell wall components.Biochemical analyses confirm that fractions enriched in root cap mucilage contain very high levels of fucose and galactose, high levels of arabinose, xylose and glucose and trace amounts of mannose. Fractions enriched in rhizodermal mucilage contain large amounts of glucose, moderate amounts of arabinose, xylose, mannose and galactose and trace levels of fucose. Isoelectric focusing and SDS-PAGE indicate that there are numerous similarities in the protein composition of materials enriched in root cap mucilages from root exudates or aqueous root surface extracts. However, specific protein bands that could be characteristic of rhizodermal mucilage are obtained using neutral saline buffer extracts. According to these biochemical data, the two-step procedure used in the present study appears to be useful for further biochemical characterization of both types of mucilages.Abbreviations BSA bovine serum albumin - BSTFA N,O-bis (trimethylsilyl)-trifluoroacetamide - DTT dithiothreitol - i. d. internal diameter - MW molecular weight - PATAg periodic acid-thiosemicarbazide-silver proteinate - PVPP polyvinylpolypyrrolidone - RE root exudates - RSE root surface extracts - TMCS trimethylchlorosilane - TMS trimethylsilyl     

Formative and proliferative cell divisions,cell differentiation,and developmental changes in the meristem of Azolla roots

The root of the water fern Azolla is a compact higher-plant organ, advantageous for studies of cell division, cell differentiation, and morphogenesis. The cell complement of A. filiculoides Lam. and A. pinnata R.Br. roots is described, and the lineages of the cell types, all derived ultimately from a tetrahedral apical cell, are characterised in terms of sites and planes of cell division within the formative zone, where the initial cells of the cell files are generated. Subsequent proliferation of the initial cells is highly specific, each cell type having its own programme of divisions prior to terminal differentiation. Both formative and proliferative divisions (but especially the former) occur in regular sequences. Two enantiomorphic forms of root develop, with the dispositions of certain types of cell correlating with the direction, dextrorse or sinistrorse, of the cell-division sequence in the apical cells. Root growth is determinate, the apical cell dividing about 55 times, and its cell-cycle duration decreasing from an initial 10 h to about 4 h during the major phase of root development. Sites of proliferation progress acropetally during aging, but do not penetrate into the zone of formative divisions. The detailed portrait of root development that was obtained is discussed with respect to genetic and epigenetic influences; quantal and non-quantal cell cycles; variation in cell-cycle durations; relationships between cell expansion and cell division: the role of the apical cell; and the limitation of the total number of mitotic cycles during root formation.     

Plant meristems: <Emphasis Type="Italic">CLAVATA3/ESR</Emphasis>-related signaling in the shoot apical meristem and the root apical meristem

The plant meristems, shoot apical meristem (SAM) and root apical meristem (RAM), are unique structures made up of a self-renewing population of undifferentiated pluripotent stem cells. The SAM produces all aerial parts of postembryonic organs, and the RAM promotes the continuous growth of roots. Even though the structures of the SAM and RAM differ, the signaling components required for stem cell maintenance seem to be relatively conserved. Both meristems utilize cell-to-cell communication to maintain proper meristematic activities and meristem organization and to coordinate new organ formation. In SAM, an essential regulatory mechanism for meristem organization is a regulatory loop between WUSCHEL (WUS) and CLAVATA (CLV), which functions in a non-cell-autonomous manner. This intercellular signaling network coordinates the development of the organization center, organ boundaries and distant organs. The CLAVATA3/ESR (CLE)-related genes produce signal peptides, which act non-cell-autonomously in the meristem regulation in SAM. In RAM, it has been suggested that a similar mechanism can regulate meristem maintenance, but these functions are largely unknown. Here, we overview the WUS–CLV signaling network for stem cell maintenance in SAM and a related mechanism in RAM maintenance. We also discuss conservation of the regulatory system for stem cells in various plant species. S. Sawa is the recipient of the BSJ Award for Young Scientist, 2007.     

Kinetics of the light-induced georeactivity of maize roots

Apical root segments of Zea mays L. cv. Orla 264 undergo some geotropic curvature in complete darkness but the curvature increases considerably if prior to geostimulation the segments are given a light pretreatment. If the light treatment is follwed by a dark treatment before the root is geostimulated the light-induced response is not changed by dark periods up to 2 h but declines with longer ones, and disappears completely after 5 h of darkness.     

Cell-lineage patterns in the shoot apical meristem of the germinating maize embryo

A fate map for the shoot apical meristem of Zea mays L. at the time of germination was constructed by examining somatic sectors (clones) induced by -rays. The shoot apical meristem produced stem, leaves, and reproductive structures above leaf 6 after germination and the analysis here concerns their formation. On 160 adult plants which had produced 17 or 18 leaves, 277 anthocyanin-deficient sectors were scored for size and position. Sectors found on the ear shoot or in the tassel most often extended into the vegetative part of the plant. Sectors ranged from one to six internodes in length and some sectors of more than one internode were observed at all positions on the plant. Single-internode sectors predominated in the basal internodes (7,8,9) while longer sectors were common in the middle and upper internodes. The apparent number of cells which gave rise to a particular internode was variable and sectors were not restricted to the lineage unit: a leaf, the internode below it, and the axillary bud and prophyll at the base of the internode. These observations established two major features of meristem activity: 1) at the time of germination the developmental fate of any cell or group of cells was not fixed, and 2) at the time of germination cells at the same location in a meristem could produce greatly different amounts of tissue in the adult plant. Consequently, the developmental fate of specific cells in the germinating meristem could only be assigned in a general way.Abbreviations ACN apparent cell number - LI, LII, LI-LII sectors restricted to the epidermis, the subepidermis, or encompassing epidermis and subepidermis - PCN progenitor cell     

Hormone interactions at the root apical meristem

Plants exhibit an amazing developmental flexibility. Plant embryogenesis results in the establishment of a simple apical-basal axis represented by apical shoot and basal root meristems. Later, during postembryonic growth, shaping of the plant body continues by the formation and activation of numerous adjacent meristems that give rise to lateral shoot branches, leaves, flowers, or lateral roots. This developmental plasticity reflects an important feature of the plant's life strategy based on the rapid reaction to different environmental stimuli, such as temperature fluctuations, availability of nutrients, light or water and response resulting in modulation of developmental programs. Plant hormones are important endogenous factors for the integration of these environmental inputs and regulation of plant development. After a period of studies focused primarily on single hormonal pathways that enabled us to understand the hormone perception and signal transduction mechanisms, it became obvious that the developmental output mediated by a single hormonal pathway is largely modified through a whole network of interactions with other hormonal pathways. In this review, we will summarize recent knowledge on hormonal networks that regulate the development and growth of root with focus on the hormonal interactions that shape the root apical meristem.     

Rapid,localized light effect on root growth in maize

A method using optical microfibers permitted localized exposure of the cap or the elongating part of growing maize (Zea mays L.) roots to white light. When the cap was illuminated, a strong and very rapid inhibition of the elongation rate of the roots was found. When the light microbeam was directed at the elongating region, the roots continued to grow at the same rate as before the illumination.     

The role of extracellular free-calcium gradients in gravitropic signalling in maize roots

Gravitropism in roots has been proposed to depend on a downward redistribution of calcium across the root cap. However, because of the many calcium-binding sites in the apoplast, redistribution might not result in a physiologically effective change in the apoplasmic calcium activity. To test whether there is such a change, we measured the effect of gravistimulation on the calcium activity of statocyte cell walls with calcium-specific microelectrodes. Such a measurement must be made on a tissue with gravity sensing cells at the surface. To obtain such a tissue, decapped maize roots (Zea mays L. cv. Golden Cross Bantam) were grown for 31 h to regenerate gravitropic sensitivity, but not root caps. The calcium activity in the apoplasm surrounding the gravity-sensing cells could then be measured. The initial pCa was 2.60 ± 0.28 (approx 2.5 mM). The calcium activity on the upper side of the root tip remained constant for 10 min after gravistimulation, then decreased 1.7-fold. On the lower side, after a similar lag the calcium activity increased 1.6-fold. Control roots, which were decapped but measured before recovering gravisensitivity (19 h), showed no change in calcium activity. To test whether this gradient is necessary for gravitropic curvature, we eliminated the calcium activity gradient during gravitropism by applying a mobile calcium-binding site (di-nitro-BAPTA; 1,2-bis(2-amino-5-nitro-phenoxy)ethane-N,N,N,N-tetraacetic acid) to the root cap; this treatment eliminated gravicurvature. A calcium gradient may be formed by proton-induced calcium desorption if there is a proton gradient. Preventing the formation of apoplastic pH gradients, using 10 and 50 mM 2-(N-morpholino)ethanesulfonic acid (Mes) buffer or 10 mM fusicoccin to stimulate proton excretion maximally, did not inhibit curvature; therefore the calcium gradient is not a secondary effect of a proton gradient. We have found a distinct and rapid differential in the apoplasmic calcium activity between the upper and lower sides of gravistimulated maize root tips which is necessary for gravitropism.Abbreviations BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - FC fusicoccin - Mes 2-(N-morpholino)ethanesulfonic acid The authors thank Phyllis Woolwine for drawing Fig. 1, Dr. Sarbjit Virk for assistance with total calcium measurements, Dr. Paul Sampson for statistical advice, and Michael Newton for developing the EM algorithm to analyze the time-series data. This work was supported by NASA grant NAGW-1394 and by a NASA Research Associateship to T.B. through NASA grant NAGW-70.