The response of apical meristems of primary roots of Vicia faba L. to colchicine treatments

The effects of 0.5% and 0.025% solutions of colchicine on the passage of cells through the mitotic cycle in apical meristems of primary roots of Vicia faba have been examined. Both treatments affected cell progression through the mitotic cycle in the same way: S and G1 were shorter, and G2 and mitosis longer, than the corresponding control values. The duration of the various phases of the mitotic cycle were similar to those reported previously for apical meristems of lateral roots though cycle time itself was longer. Recovery of root proliferating tissues from colchicine-induced inhibition of growth is correlated with the presence of quiescent cells. Meristems which have no quiescent cells do not recover from eolchicine treatment, while meristems which contain many quiescent cells recover faster than those which contain few. The growth fraction and the proportion of proliferating cells with a short cycle time are linearly related to the duration of the S period in root meristems.     

Thymidine kinase and thymidylate synthetase in meristems of roots ofVicia faba

Summary Labelling and inhibitor studies on LP, SP, and lateral root apical meristems have demonstrated the presence of the enzymes TdR kinase and TMP synthetase in active form in all three proliferating tissues. A permeability barrier in the epidermis and cortex of the primary root and also the fluid filled cavity between LP and the cortex prevent much exogenously supplied deoxynucleoside from reaching these cells. Values have been obtained for the duration of G₂ in both apical meristems of lateral roots and SP, and, by using data previously reported elsewhere, the durations of all phases of the mitotic cycle in SP have been calculated.     

The Difference Between Open and Closed Meristems

An open and a closed root meristem have been compared by investigatingthe cell kinetics of small regions of the apices of Helianthusand Zea. The cells of the stelar pole are quiescent in both and thereis no exchange of cells between stele and cortex or stele andcap. The immediately distal cells in the closed meristem (Zea)are also quiescent and the few divisions that do occur can betransverse or longitudinal. In the open meristem (Helianthus)these cells are not quiescent, but they go out of cycle transiently,prolonging the potential cell-doubling time. Their divisionsare transverse. It is a consequence of these differences thatclosed meristems form root caps discrete from the cortex whereasopen meristems force instability in the boundary between theperipheral part of the cap and the cortex. Another consequencein roots with open meristems is a succession of columella complexestransversely displaced from each other by the state of fluxin the meristem during the non-cycling phase of the proximaltier of cells, those immediately distal to the stelar pole. The results are discussed in relation to the ontogenetic onsetof quiescence and the evidence for switches between open andclosed operation of meristems. meristem, root apex, Helianthus annuus, Zea mays L.     

The Response of Root Meristems to Colchicine and Indol-3yl-acetic acid in Vicia faba L.

The effects of colchicine and IAA treatments on mitotic activityin various root proliferating tissues have been determined.Lateral root primordia were not affected by IAA, though 24 hfollowing treatment mitotic activity was severely inhibitedin the apical meristems of 1-cm-long attached lateral rootsand primary roots. Primordia were also less sensitive to colchicinetreatment than root apical meristems. Thus telophase figureswere present in the former meristems 3 h following treatment,but not in the latter. Primordia and apical meristems respondedto the same extent, however, to the colchicine-induced increasein number of cells in metaphase, anaphase, and telophase, 3h after treatment began. The apparent difference between largeprimordia and root apical meristems in this respect was dueto the failure of colchicine to penetrate the cells of the formerproliferating tissues as rapidly as the latter. IAA was foundto prevent the increased MI found 24 h following colchicinetreatment only in those meristems where IAA inhibited mitoticactivity at this time. IAA treatments, either alone or withcolchicine, were also found to maintain mitotic activity in1-cm-long lateral roots which were excised from the primaryroots 24 h previously. In such laterals which were not treatedwith IAA, MI was zero at 24 h. It is concluded from the datareported in this paper that, during the development of rootapical meristems, changes take place in the response of cellsto factors affecting mitotic activity.     

Some effects of 2,4,5-trichlorophenoxyacetic acid on the mitotic cycle of lateral root apical meristems of Vicia faba

Roots of Vicia faba were given a one hour pulse label with. ³H-TdR (1 C/ml), either before or after a three hour treatment with a 10^–5 M solution of 2,4,5-trichlorophenoxyacetic acid (TCPA). The durations of the various phases of the mitotic cycle were derived from labeled prophase curves, prepared from autoradiographs of lateral root apical meristems. — TCPA was found to lengthen the duration of the mitotic cycle, primarily because it extended the duration of the period of DNA synthesis (S), though post-synthetic interphase (G₂) was also longer. No measurements could be made with respect to the duration of presynthetic interphase (G₁), because of rapid changes in the lengths of the G₂ and S periods following treatment. — As well as extending the duration of S, TCPA treatment also resulted in at least an initial increase in the rate of DNA synthesis and a decrease in the actual number of cells in S. These results have been discussed with respect to the control of the organization of the root apical meristem.Supported by a grant from the Assistant Professor Research Fund of the University of Missouri.     

CELL DIVISION KINETICS IN THE SHOOT APICAL MERISTEM OF CERATOPTERIS THALICTROIDES BRONG. WITH SPECIAL REFERENCE TO THE APICAL CELL

The duration of mitosis and the cell cycle were determined for defined cell populations of the shoot apical meristem of Ceratopteris thalictroides Brong. by using the colchicine-induced metaphase accumulation technique. The results indicate that the apical cell is mitotically active and cycles at an apparently greater frequency than the cells of subjacent populations. Duration of mitosis was similar for all cells of the meristem. These results are correlated with mitotic indices of control apices, the geometry of the apex, and the mean number of cells in the meristem. Shoot apices from adult plants were examined to determine mitotic indices within the meristem; mitotic activity was again noted for the apical cell. These results contradict recent proposals that the pteridophyte apical cell serves as a unicellular quiescent center which lacks histogenic potential and offer experimental support for the classical concept of apical cell function in those fern shoot meristems which terminate in a single apical cell.     

Organelle DNA Synthesis in the Quiescent centre of Arabidopsis thaliana (Col.)

The behaviour of cell nuclei and organelle nucleoids (organellenuclei) was studied in the root apical meristem of 3-d-old seedlingsof Arabidopsis thaliana (Col.). Samples were embedded in Technovit7100 resin, cut into thin sections and stained with 4'-6-diamidino-2-phenylindole(DAPI) for observation of DNA. DNA synthesis in cell nucleiand organelle nucleoids was investigated using the incorporationof [³H] thymidine or 5-bromo-2'-deoxyuridine (BrdU). Incorporated[³H] thymidine and BrdU were detected by microautoradiographyor immunofiuorescence microscopy, respectively. Central cellsand cells just above the central cells of the quiescent centre(QC) showed an extremely low activity of DNA synthesis. However,DNA synthesis occurred in at least one organelle nucleoid ofall cells in the QC within 24 h. This suggests the cells inthe QC are quiescent with regard to nuclear DNA synthesis, butnot with regard to the organelle nucleoids. Key words: Arabidopsis thaliana, quiescent centre, root apical meristem, mitochondrial nucleoid (nuclei), plastid nucleoid (nuclei)     

The Quiescent Centre and Rates of Mitosis in the Root Meristem of Allium sativum

The root apices of Allium sativum have been examined by continuous-and pulse-labelling with tritiated thymidine and by colchicinetreatment to measure the time parameters of the mitotic cyclein various parts of the meristem. There is a quiescent centre of 30–50 cells whose averagerate of mitosis is low because the G₁ period is extended toabout 140 h compared with about 4 h in the othe regions of themeristem. The stele just above the quiescent centre and at 200microns above it and the cap initials just below the quiescentcentre are very similar in their mitotic cycles, the total lengthsof which are about 30 h of which nearly half is taken up byDNA synthesis. Allium thus differs from Zea in having root capinitials whose mitotic cycle is not telescoped by the eliminationof the G₁ phase. These facts are discussed in relation to theradiosensitivity of the meristem.     

The effect of Lead on early stages ofPhaseolus vulgaris L. growthin vitro conditions

Lead chloride (10^-5 M) inhibited the growth of the main root, the duration of development, the number and growth of lateral roots, primary and trifoliate leaves, and also the mitotic index in root apical meristems. Lead strongly inhibited root growth rate, mainly by reducing the number of dividing cells. Other mechanisms of this inhibition are discussed.     

The Nuclear Endoreduplication Cycle in Metaxylem Cells of Primary Roots of Zea mays L.

The nuclear DNA content of metaxylem cells in roots of Zea mayscv. Golden Bantam reaches 16C or 32C by successive rounds ofDNA endoreduplication. Each phase of endoreduplication (endo-S)is separated by a non-DNA synthetic phase (endo-G). These phasesseem to occur in zones at fixed distances from the root tip.The duration of the phases in two of the endoreduplication cycles(4C–8C, 8C–16C) has been estimated in two ways.The first makes use of the rate of movement of cells throughthe positions along the root where the different phases of thecycle are occurring, the second uses labelling with methyl-[³H]thymidineand autoradiography. Both methods indicate that the endo-S phaseswhich cause the nuclear DNA content to rise from 4C to 8C andfrom 8C to 16C last 8–10 h, and that the intervening endo-Gphase lasts 8–12 h. DNA endoreduplication keeps pace withthe increase of nuclear volume; cell volume increases at a morerapid rate, however. Comparison of the endoreduplication cyclein the metaxylem with the mitotic cycle in the adjoining filesof parenchyma cells shows that the mitotic cells complete theircycle more slowly. DNA synthesis, endoreduplication cycle, mitotic cycle, root apex, Zea mays     

Changes in the Duration of the Mitotic Cycle Induced by Colchicine and Indol-3yl-acetic Acid in Vicia faba Roots

Cells of root meristems of Vicia faba were labelled with tritiatedthymidine and treated with colchicine or IAA or both. The effectsof these compounds on the duration of the mitotic cycle andits constituent phases have been determined using the labelledmitoses wave method of Quastler and Sherman. Colchicine shortensthe mitotic cycle of the cells in interphase at the time oftreatment; it appears to stimulate cells in G1 or early S tocomplete interphase faster than untreated cells. The affectedcells arrive at mitosis 9–12 h after the beginning oftreatment and contribute to the increase in mitotic index seenafter treatment with colchicine. Treatment with IAA did notaffect cells in G2 but it delayed cells in S; this results ina temporary fall in M.I. The effect of IAA in prolonging interphasewas also seen in roots treated with colchicine and IAA; thetetraploid cells induced by colchicine take longer to reachmetaphase than cells treated only with colchicine. The resultssuggest that colchicine and IAA affect different phases of thecell cycle.     

Proliferating and Quiescent Cells in the Apical Meristem of Elongating Lateral Roots of Vicia faba L.

Whole root systems of Vicla faba were continuously exposed to³H-TdR for periods of up to 72h, following which LI was determinedin the cap initials, in the quiescent centre or in that partof the meristem in which a quiescent centre will develop, andin the stele and in the cortex-epidermis at intervals alongthe apical 800 µm basal to the junction between the capinitials and the rest of the meristem, in newly-emerged (NE),0.2 and 4.0 cm long lateral roots, after each exposure period.Cell doubling time (Td), mean cycling time (Tc) and the sizeof the growth fraction (GF) were then calculated for each partof the meristem investigated in each batch of roots, from thecurves recording increase in labelling index (LI) with increasein duration of the period of exposure to ³H-TdR and from therate of increase in LI over the initial l-12h labelling period.Since it is extremely difficult to eliminate all sources oferror in calculating GF from the values obtained for LI in continuouslabelling experiments, it is emphasized that the values of GFreported in the present paper may not be totally accurate. Thisis also true of the results obtained for Tc as Tc was derivedfrom the product of the corresponding values for Td and GF. Cell doubling time and mean cycling time were both longer inthe cells forming the quiescent centre in the 0.2 and 40 cmlong roots than in any other part of the apical meristem examined.The size of the GF was found to decrease basally along the steleand the cortex-epidermis from the most apical to the most basalsegment examined in the NE, while Td increased in duration.Similar changes took place along the stele of the 0.2 cm longlaterals, but not in the cortex-epidermis of these roots or,to any great extent, in any of the tissues examined in the 4.0cm secondary roots. No consistent trend was apparent in theduration of Tc basally along either tissue examined in theseroots. It was concluded from these results, and from supportingdata in the literature, that, as the laterals elongated fromNE to 4.0 cm, the apical meristem increased in length.     

β-glucuronidase as a marker for clonal analysis of tomato lateral roots

In higher plants, the root-shoot axis established during embryogenesis is extended and modified by the development of primary and lateral apical meristems. While the structure of several shoot apical meristems has been deduced by combining histological studies with clonal analysis, the application of this approach to root apical meristems has been limited by a lack of visible genetic markers. We have tested the feasibility of using a synthetic gene consisting of the maize transposable elementActivator (Ac) inserted between a 35S CaMV promoter and the coding region of a -glucuronidase (GUS) reporter gene as a means of marking cell lineages in roots. The GUS gene was activated in individual cells byAc excision, and the resulting sectors of GUS-expressing cells were detected with the histochemical stain X-Gluc. Sectors in lateral roots originated from bothAc excision in meristematic cells and from parent root sectors that bisect the founder cell population for the lateral root initial. Analysis of root tip sectors confirmed that the root cap, and root proper have separate initials. Large sectors in the body of the lateral root encompassed both cortex and vascular tissues. The number of primary initial cells predicted from the size and arrangement of the sectors observed ranged from two to four and appeared to vary between roots. We conclude that transposon-based clonal analysis using GUS expression as a genetic marker is an effective approach for deducing the functional organization of root apical meristems.     

Changes in the Chromosome Complements of Cells of Vicia faba Roots following Irradiation

1. Primary roots of Vicia faba were irradiate after one day'sgermination. The X-ray dose was 620 r 2. Cells with changed chromosome complements appeared in lateralroots. It is inferred that these cells are descended from cellsdamaged by irradiation; chromosome breakage was seen in thefirst mitoses in the irradiated roots but was not scored. 3. Six types of chromosomal rearrangements were found. One typehad been reported previously in primary roots of V. faba afterX-raying. 4. The new complements appear to be stable because they occurseveral cells of a lateral. Affected laterals could containup to five new complements. The primordium of such a lateralmust have consisted of five changed and at least one normalcell. It had previously been suggested that a root of Viciahad three or four initial cells. 5. The survival of the cells with changed complements is notdue solely to successful competition with, not to support by,normal cells, otherwise other types of changes should also havebeen found. 6. It is suggest that the changes found were perpetuated becausethey were induced in initial cells of the primary root, andthat cells in which they were present beca,e part of the primordialand continued as initial cells in apical meristems of lateralroots. 7. Apical cells appear to have a lower sensitivity than othermeristematic cells, so providing, after treatment, a reservoirof normal cells from which regeneration can occur.     

The Expression Pattern of the Gene for NPK1 Protein Kinase Related to Mitogen-Activated Protein Kinase Kinase Kinase (MAPKKK) in a Tobacco Plant: Correlation with Cell Proliferation

Mitogen-activated protein kinase (MAPK) cascades consist ofmembers of three families of protein kinases: the MAPK family,the MAPK kinase family, and the MAPK kinase kinase (MAPKKK)family. Some of these cascades have been shown to play centralroles in the transmission of signals that control various cellularprocesses including cell proliferation. Protein kinase NPK1is a structural and functional tobacco homologue of MAPKKK,but its physiological function is yet unknown. In the presentstudy, we have investigated sites of expression of the NPK1gene in a tobacco plant and developmental and physiologicalcontrols of this expression. After germination, expression ofNPK1 was first detected in tips of a radicle and cotyledons,then in shoot and root apical meristems, surrounding tissuesof the apical meristems, primordia of lateral roots, and youngdeveloping organs. No expression was, however, observed in matureorgans. Incubation of discs from mature leaves of tobacco withboth auxin and cytokinin induced NPK1 expression before thedivision of cells. It was also induced at early stages of thedevelopment of primordia of lateral roots and adventitious roots.Thus, NPK1 expression appears to be tightly correlated withcell division or division competence. Even when an inhibitorof DNA synthesis was added during the germination or the inductionof lateral roots by auxin, NPK1 expression was detected. Theseresults showed that the NPK1 expression precedes DNA replication.We propose that NPK1 participates in a process involving thedivision of plant cells. (Received January 26, 1998; Accepted April 9, 1998)     

Exit from the Mitotic Cycle in Root Meristems of Zea mays L.

The choice between two modes of exit from the mitotic cycleat the margins of meristems has been made easier by surveyingthe range of the numbers of cell contacts between contiguousfiles in root apices of Zea mays L. The range shows that somecells must go out of cycle while others remain in cycle forat least three further generations. The view that cycling endsby a fall in the proliferative fraction is supported by theexistence of pulse-labelled telophases in the proximal regionof the menstem. These are most likely due to acceleration ofthe mitotic cycle which has to be contrasted with decelerationof the overall rate of cell proliferation. The work is discussedin relation to patterns of cycling in the different tissuesof the apex. mitotic cycle, cell size, meristem, proliferative fraction, Zea mays L, maize     

Mitotic cycle kinetics of root meristems of isolated barley embryos and intact seedlings. Labelling of nuclei by3H- thymidine and its cytogenetic consequences

The duration of the mitotic cycle and its individual phases was estimated in root meristems of isolated barley embryos and intact barley seedlings by means of pulse labelling with³H-thymidine and construction of labelled mitoses curve. The duration of the whole mitotic cycle in the cell population of root meristems of isolated barley embryos cultivated in the aerated liquid complete medium is 12.2 h. The mitotic cycle time of root meristems of intact barley seedlings, oultived in Petri dishes on wet blotting paper is 9.2 h. Most of root meristem cells belong to the fraction of rapidly proliferating cells, but this fraction exerts a high degree of variability by itself. Pulse treatment by³H-thymidine in our experimental conditions (74 kBq ml^-1 - or 2 μCi ml^-1, exposure 0.5 h) did not induoe any chromosomal aberrations in unlabelled cells and only a very low frequency of chromosomal aberrations in labelled cells. Measuring the cell population kinetics by pulse labelling with³H-thymidine can be used simultaneously with the study of induction of ohromosomal aberrations by mutagens.     

Mitotic Activities and Levels of Nuclear DNA in the Apical Meristem of Silene armeria (strain SI.2) Following Application of Gibberellin A3

Strain S1.2 of Silene armeria was grown under an 8h-photoperiodand treated with GA₃ every day for 20 days. This growth substancecaused stem elongation, but no flowering in this long-day plant.Changes in the mitotic index and DNA content of cells in thevarious zones of the apical meristem before, during and afterGA₃ treatment were described. Mitotic activity and increasein the proportion of nuclei at the 4C level (S+G₂ phase of thecell cycle) were strongly stimulated in the rib-meristem, andto a lesser extent in the lateral zone, but not in the axialzone. This stimulation of apical activity reached a peak aftertwo GA₃ treatments and then declined gradually, so that after20 days the activity in GA₃-treated meristems was lower thanthat in untreated controls; at this point most cells were inthe G₁ phase. When the GA₃ treatment was discontinued, there was a gradualincrease in the mitotic activity which ultimately reached thesame level as that in controls. Stem elongation ceased and leavesformed aerial rosettes. It is concluded that in vegetative plants of strain S1.2 ofSilene armeria GA₃ acts mainly on the rib-meristem cells whichresults in stem elongation. Lack of response in the axial cellsexplains why GA₃ fails to induce flowering in this strain ofSilene armeria. (Received June 18, 1983; Accepted August 3, 1983)     

The extent and differences in mitotic activity of the root tip ofVicia faba L.

Mitotic activity does not stop for different meristematic cells of the root apex at the same distance from the initials. The differences are connected with the functional heterogeneity of the apical meristem of the root. The arrangement of vascular bundles,i.e. the alternation of independent xylem and phloem groups, is of major importance. In broad bean roots, the protophloem sieve elements stop dividing first. The centre of the stelei. e. late metaxylem elements stop dividing next. Division in the stele gradually ceases centrifugally, while it ceases centripetally in the peripheral part of the root. The cylindrical region with prolonged cell division includes internal layers of the cortex including endodermis, pericycle and adjoining cells of the stele. Proximally apical meristem is reduced to isolated strands of cells adjacent to the protoxylem poles. Pericycle cells stop dividing last at a distance of approx. 9–10 mm from the initials. The number of the division cycles is limited and is specific for individual cell types. Epidermal and cortical cells divide in broad bean roots transversely approximately seven times, cells of late metaxylem approximately five times. Root apical meristem is an asynchronous cell population with a different duration of the mitotic cycle. We determined local variations in the duration of the mitotic cycle in the apical meristem of broad bean root by means of colchicine-induced polyploidy. The cells of the quiescent centre had the longest mitotic cycle after colchicine treatment. The region of the proper root adjacent to the quiescent centre was mixoploid (2n and 4n). Isolated cells with a long cycle occurred also in the cortex and in the central cylinder. Cells with a division cycle of 18h were found in the root cap, in the epidermis, in the cortex and in the central cylinder. Relatively numerous cells with the shortest division cycle, approx. 12 h, occurred farther of the quiescent centre in the epidermis, in the cortex, in the pericycle, and in adjacent layers of the stele through-out the entire meristematic region. The results derived from the analysis of the apical meristem are discussed in connection with the ontogenesis of different types of cells taking part in the primary structure of the root.     

Some Cytological Changes accompanying the Regeneration of Meristematic Activity at the Apex of Decapitated Roots of Vicia faba L.

The changes that took place in mitotic index (MI), labellingindex (LI) and the relative proportions of interphase nucleiwith different amounts of DNA have been investigated duringthe regeneration of meristematic activity at the apex of rootsof Vicia faba over the 144 h period following removal of thecap and apical mm of the meristem. Measurements were also madeof the corresponding changes that took place as cells were displacedbasally along the root from the apex over the experimental period.In both parts of the root, MI and the relative proportions ofnuclei with different DNA contents changed from levels similarto those at the apex of the controls at the start and end ofthe experiment to levels resembling those found in more matureparts of the root at 24 and 48 h. In contrast to these results,LI declined over the experimental period. These cytologicalchanges were aresult of the development of lateral root primordiain both the apical 2 mm of the decapitated roots and as cellswere displaced out of the meristem into more basal parts ofthe root. It was concluded that the events leading to the regenerationof meristematic activity at the apex of roots from which thecap and apical mm of the meristem were removed, are no differentfrom those which result in lateral formation as cells are displacedbasally along the primary root from the apex, and they takeplace over the same time interval in both systems.