The Response of Primordial Cells of Vicia faba to Colchicine

Whole root systems of Vicia faba were treated with 0.025 percent colchicine for 3 h. Meristems of roots and developing primordiawere examined immediately after treatment and 24 and 48 h later.Large primordia, i.e. those primordia that would emerge as lateralroots within approximately 24 h, are induced to form tetraploidcells. However, unlike lateral roots, they do not form a typicalc-tumour, their longitudinal growth is not inhibited, and theirmitotic index does not fall. Furthermore, the tetraploid cellsinduced in large primordia are not maintained when the primordiaemerge as lateral roots. Since the response of the very largestprimordia to a 3-h treatment with 0.025 per cent colchicineis similar in every way to that of lateral roots, it appearsthat the cells of primordia acquire the characteristics of lateralroot meristematic cells before the primordia emerge. From theevidence that induced tetraploid cells fail to survive as aprimordium grows out to form a lateral, it also seems that astructural reorganization accompanies the physiological reorganizationthat occurs as a primordium emerges.     

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.     

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.     

Studies on induced changes in growth patterns and polarity in roots of Vicia faba L.

Summary Roots of Vicia faba were treated with colchicine (0.025%), or IAA (4.7×10^-6 M), or both, for 3 hours and fixed at various intervals over the following 11 days. The axis of spindle orientation and the distribution of mitotic figures, lateral root primordia and xylem vessel elements was examined in the apical 10 mm of median longitudinal sections of these roots.No effect of IAA was found on the orientation of the spindle. However, evidence was obtained indicating that the systems controlling the polarity of cell division and cell expansion differ in some way.The number of lateral root primordia formed was greater in roots treated with IAA or colchicine than in control roots. These primordia were always initiated adjacent to a xylem vessel. Thus, no primordium was closer to the apex than the most apical xylem vessel, suggesting that an endogenous factor involved in primordia initiation is transported in the xylem. The primordia which develop after colchicine treatment grow out as lateral roots; this is in contrast with those which form after IAA treatment and which do not undergo elongation. These results, which it must be emphasized apply only to the apical 1 cm of treated roots, indicate that lateral root primordia become sensitive to IAA at a certain stage in their development. Exogenous IAA acts as an inhibitor.The new meristem, which forms in the primary root apex after colchicine treatment, contains both diploid and polyploid cells, i.e. it was formed from cells that were unaffected and from cells that were affected by colchicine. Following colchicine treatment the size of the meristem shrinks and this can be prevented by treatment with IAA. This and other evidence presented here, suggests that IAA is a factor involved in the control of the size of the apical meristem in normal roots.     

Auxin Induced Lateral Root Formation in Chicory

The supply of auxins [2,4-dichlorophenoxy acetic acid (2,4D),indole-3 acetic acid (1AA) and -naphthaleneacetic acid (NAA)]to excised chicory roots induced the formation of lateral rootmeristems mainly located close to the pre-existing apical rootmeristem. Lateral root growth induced in non-excised roots requiredhigher auxin concentrations. Inhibition of root elongation andconcomittant enlargement of the apices was also observed. SupplyingIAA induced the formation of lateral meristems earlier thanNAA, but subsequently favoured root elongation. Conversely,in the presence of 2,4D, reactivation of pericycle cells wasvery intense, but conversion of primordia to laterals was inhibited.Regardless of the auxin used, the responsive area in which lateralmeristems appeared was located a maximum of 4 mm away from theapical meristem. This region remained devoid of any lateralroot formation under control conditions. Pericycle cells oppositethe xylem poles in the diarch stele regained meristematic activityand divided transversally, giving rise to shorter cells. Thesecells subsequently divided periclinally, forming pairs of cellson the same transverse level. The root primordium extruded throughcortical cells and was surrounded by a lacuna formed to thedetriment of cortical cells.Copyright 1998 Annals of BotanyCompany Auxins,Cichorium intybus, chicory, lateral root, root elongation.     

Cell Population Studies in Developing Root Primordia

The proliferation of polyploid and diploid cells was followedduring the development of lateral root primordia of Vicia faba.Presumptive primordial cells were treated with weak solutionsof colchicine, 0.003, 0.006, and 0.0125 per cent, for 3 h. Somecells are induced to become polyploid and they form a markedsub-population. The relative frequencies of diploid and polyploidcells were determined from the time small primordia were presenttill lateral roots were fully emerged. These frequencies havebeen found to change in a regular fashion and the changes reflectdifferent levels of mitotic activity in different parts of aprimordia as it develops. Polyploid cells occupy the centralcore of the primordia and they divide actively except for theperiod just before lateral root emergence. Coincident with thistemporary quiescence of the core cells, the peripheral cells,which remain diploid after treatment with the weaker solutionsof colchicine, continue to divide. These changes in mitoticactivity of the central and peripheral cells are shown bothin the changes in the relative frequencies of polyploid anddiploid mitoses as a primordium grows and in changes in mitoticindex. A microspectrophotometric study indicates that the centralcells are held, temporarily in the G₁ phase of the cell cycle.     

Cortical Cell Breakdown and Lateral Root Primordium Development in Vicia faba L

The effect of the formation of a cavity in the cortex of theprimary root of Vicia faba adjacent to lateral root primordiaon root development has been investigated. Premature exposureof such primordia to the external medium by removing the overlyingtissues of the primary root has no effect on primordium developmentif that primordium was within 48 h of emerging as a lateralroot. Similar exposure of primordia which were at an earlierstage of development and consisted of between 3400 and 7000cells resulted in the generation of a stationary phase, withmost of the nuclei arrested in G1 (presynthetic interphase),48–72 h after exposure began, followed by nuclear degenerationby 96 h. Since no mature vascular tissue was found in theseprimordia until after they emerged as secondary roots, all ofthe nutrients necessary for the maintenance of cell proliferationin these meristems must reach them by simple diffusion fromthe surrounding medium. A preliminary analysis of the liquidcontents of the cavity next to developing primordia demonstratesit to be rich in carbohydrates and it is clear, from the resultsreported in this paper, that cell proliferation in primordia,consisting of a mean number of 5400 cells, is largely dependenton the substances present in the cavity fluid, although somematerials reach the primordium by diffusion from the cells ofthe primary root to which the primordium remains attached.     

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)     

Ethylene–auxin interactions regulate lateral root initiation and emergence in Arabidopsis thaliana

Plant root systems display considerable plasticity in response to endogenous and environmental signals. Auxin stimulates pericycle cells within elongating primary roots to enter de novo organogenesis, leading to the establishment of new lateral root meristems. Crosstalk between auxin and ethylene in root elongation has been demonstrated, but interactions between these hormones in root branching are not well characterized. We find that enhanced ethylene synthesis, resulting from the application of low concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), promotes the initiation of lateral root primordia. Treatment with higher doses of ACC strongly inhibits the ability of pericycle cells to initiate new lateral root primordia, but promotes the emergence of existing lateral root primordia: behaviour that is also seen in the eto1 mutation. These effects are correlated with decreased pericycle cell length and increased lateral root primordia cell width. When auxin is applied simultaneously with ACC, ACC is unable to prevent the auxin stimulation of lateral root formation in the root tissues formed prior to ACC exposure. However, in root tissues formed after transfer to ACC, in which elongation is reduced, auxin does not rescue the ethylene inhibition of primordia initiation, but instead increases it by several fold. Mutations that block auxin responses, slr1 and arf7 arf19, render initiation of lateral root primordia insensitive to the promoting effect of low ethylene levels, and mutations that inhibit ethylene-stimulated auxin biosynthesis, wei2 and wei7 , reduce the inhibitory effect of higher ethylene levels, consistent with ethylene regulating root branching through interactions with auxin.     

Lateral Root Development in Attached and Excised Roots of Zea mays L. Cultivated in the Presence or Absence of Indol-3-yl Acetic Acid

The initiation of lateral root primordia and their subsequentemergence as secondary roots have been examined in attachedand excised roots of Zea mays grown in the presence or absenceof indol-3-yl acetic acid (IAA). Exposure to IAA enhanced anlageinception in both batches of roots. In the attached roots, theIAA-induced stimulation of primordium initiation was followedby a similar increase in lateral emergence. IAA treatment, however,had no effect on the number of laterals produced, per centimetreof root, in the excised primaries. Thus, exposure to IAA didnot directly enhance lateral emergence in the attached rootsnor did it stimulate such emergence in the excised ones. Nocorrelation was found between proliferative activity in themeristem at the apex of the primary or the rate of root elongationon the one hand, and either the number of primordia initiated,or the number of laterals produced, per centimetre of primary,on the other. Zea mays, maize, root, primordium, lateral, indol-3-yl acetic acid, meristematic activity     

The Relationship Between Lateral-Root Distribution and Endodermis and Pericycle Cell Length in Allium cepa L. Adventitious Roots

Endodermis and pericycle cell lengths were measured in intactand decapitated adventitious roots of Allium cepa L. Decapitationhad no effect on cell length in mature portions of the root,although it affected more immature cells, impeding normal elongation.Cell length shows a characteristic pattern in different zonesof the adventitious root: cells in the medial region were moremarkedly elongated. The number of lateral root primordia wasalso determined in different zones of the adventitious root.The possible relationship between lateral root distributionpattern and cell length in the endodermis and pericycle is discussed. Allium cepa, onion, endodermis, pericycle, lateral root, cell length     

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.     

Site, Scale and Time-course for Adjustments in Lateral Root Initiation in Wheat Following Changes in C and N Supply

The pattern of lateral root initiation in seminal roots of wheat(Triticum aestivumL. cv. Alexandria) and the location, scaleand time-course for adjustments in initiation were studied afterchanges in C and N supply. Macroscopically visible primordiaappeared in a non-acropetal sequence with the frequency (numberper unit length) increasing with distance behind the main rootapex to a maximum at 40–50 mm behind the root tip. Pruningthe root system to a single seminal axis increased the primordiafrequency by 23% within 15 h. After longer periods, the effectof root-pruning was greater. The enhanced primordia frequencywas first observed in tissue located 0–10 mm behind theapex at the start of treatment. Feeding glucose (50 mM) alsoincreased primordia frequency within 15 h, but to a greaterextent, and here additional primordia were initiated in tissuelocated 0–10and10–20 mm behind the apex at the startof treatment. Withdrawing NO₃^-from one part of a split-rootsystem, whilst maintaining the supply to the other, reducedprimordia frequency in the non-fed roots and, in some cases,a compensatory increase in the NO₃^--fed roots was observed.The location and scale of the adjustments were similar to thosefound with root-pruning and glucose-feeding, but were slightlyslower to appear. In spite of some differences in detail, therewas a broad similarity in site, scale and time-course for adjustmentsin lateral root initiation with these treatments, which is consistentwith the operation of a common mechanism. Whenever an increasein primordia frequency was observed, it was associated withan increase in the ethanol-soluble sugar content of the tissue.However, the reduction in frequency in NO₃^--deprived roots wasalso accompanied by an increase in sugar content. There wasno consistent relationship between total N content of the tissueand primordia frequency, but there was between primordia frequencyand the rate of net NO₃^-uptake. The possible mechanisms controllinglateral root initiation are discussed. Compensatory growth; correlative growth; glucose; initiation; lateral root; nitrate; primordium; split-root; Triticum aestivum; wheat     

Root Growth Inhibitors from Root Cap and Root Meristem of Zea mays L.

A micro-assay based on the growth inhibition of root segmentsof the seminal roots of Zea mays has been used to investigatethe root-growth-inhibiting substances in root caps and meristemsrespectively of the roots of Zea mays. This micro-assay is sensitiveto 50 pg of IAA or less. Paper chromatography of the acid fractionof methanolic extracts shows the presence of one main inhibitorin root caps and a different main inhibitor in root meristems.Neither is IAA, whose presence in meristems is sometimes indicatedby small inhibitions (or stimulations) at the characteristicRf of IAA. A Commelina leaf-epidermis assay shows the presenceof one stomata-closing ABA-like substance in root caps and onein meristems, one corresponding in Rf to the main root-growthinhibitor from the root cap. The implications of these findingsfor the geotropic responses of roots is briefly discussed.     

Lateral Root Anlage Development in Excised Roots of Vicia faba L., Pisum sativum L., Zea mays L. and Phaseolus vulgaris L.

The development of lateral root primordia has been investigatedin excised roots of Vicia faba, Pisum sativum, Zea mays andPhaseolus vulgaris cultured in White's medium supplemented with2 per cent sucrose and compared with previously published dataon such development in primaries of the corresponding intactplants (control roots). Primordia were produced in each batchof excised roots over the 6 day culture period but at a lowerrate (number day^–1) than in the controls. Such primordia in cultured roots of Zea and Phaseolus completedtheir development and grew out as lateral roots over a periodsimilar in length to that found in the controls, but with acell number of only about 33 per cent of that attained at thetime of secondary emergence in the primaries of the latter roots.These lower cell numbers were at least partly a reflection ofincreases in mean cell doubling time over the period of anlagedevelopment investigated in the excised roots relative to thecorresponding values found in the controls. Primordia initiated in excised roots of Pisum and Vicia didnot complete their development in culture, i.e. no lateral rootsemerged and arrest took place with cell numbers of only 37 (Pisum)and 17 (Vicia) per cent of the numbers determined at the timeof secondary root emergence in the controls. Such arrested primordiahad few nuclei in S and none in mitosis. Moreover, at leastin Pisum, the frequency distribution of the relative DNA contentof the nuclei in the latter primordia approximated that foundin the apical meristem of primary roots following the establishmentof the stationary phase under conditions of carbohydrate starvation. It has also been demonstrated in the course of these investigationsthat lateral root primordium development in all four speciesis at least biphasic and possibly triphasic. Vicia faba L., broad bean, Pisum sativum L., garden pea, Zea mays L., maize, Phaseolus vulgaris L., dwarf bean, root primordia, anlage, cell doubling time, lateral root emergence     

The Control of Lateral Root Development in Cultured Pea Seedlings. III. Spacing Intervals

The spacing of lateral root primordia in the primary root of Pisum sativum (cv. Alaska) seedlings is influenced by both predetermined lateral root initiation sites in the embryonic radicle and by factors present during seedling growth. When pea seeds were germinated in the presence of the mitotic inhibitor, colchicine, the triarch radicle produced three ranks of primordiomorphs indicating sites of embryonic lateral root primordia. The number of primordiomorphs was not the same along the three xylem strands in the radicle. Normally germinated seedling roots (5 days old) also showed a different number of lateral root primordia associated with the three strands. In both cases, the strand with the greatest number of primordia (or primordiomorphs) was associated with a cotyledonary trace. This indicated a possible role for the cotyledons in setting the pattern of lateral root distribution during radicle development. The spacing of lateral root primordia could be altered by the application of growth regulators. Seedling root tips (2 mm) were removed (? rt) and replaced with indoleacetic acid (+IAA), and in some instances seedlings were also treated with the auxin transport inhibitor, 3,3a-dihydro-2-(p-methoxyphenyl)-8H-pyrazolo[5, 1-α]isoindol-8-one (+DPX). In the growth regulator treatments, primary root elongation was inhibited, a greater number of lateral root primordia were initiated compared to controls, and the spacing intervals between primordia were greatly reduced. The — rt, +IAA, +DPX-treatment resulted in the closest possible spacing intervals (av. 0.4 ? 0.6 mm), but resulted in fused or fasciated laterals. The — rt, + IAA-treatment produced the shortest spacing intervals which resulted in “normal” lateral roots (0.8 ? 1.1 mm).     

A greenprint for growth: signalling the pattern of proliferation

The shoot and root apical meristems (SAM and RAM, respectively) of plants serve both as sites of cell division and as stem cell niches. The SAM is also responsible for the initiation of new leaves, whereas the analogous process of lateral root initiation occurs in the pericycle, a specialized layer of cells that retains organogenic potential within an otherwise non-dividing region of the root. A picture is emerging of how cell division, growth, and differentiation are coordinated in the meristems and lateral organ primordia of plants. This is starting to reveal striking parallels between the control of stem cell maintenance in both shoots and roots, and to provide information on how signalling from developmental processes and the environment impact on cell behaviour within meristems.     

The effects of diclofop-methyl on root growth of wild oat

Diclofop-methyl severely reduced the growth of seminal roots of wild oat ( Avena fatua L.) when applied in hydroponics at 0.01 and 0.05 μ M . Lateral roots emerged closer to the seminal root apex than in the controls, but coronal root number and length were unaffected at 0.01 μ M . However, doses of 0.05 to 0.1 μ M induced more but shorter coronal roots to emerge than for controls. At 1 μ M the number and length of coronal roots were less than for controls. Root-applied diclofop-methyl at 1 μ M inhibited emerging second leaf growth to the same extent as a foliar dip in 1 μ M diclofop-methyl without causing chlorosis as foliar treatment does. Because of limited basipetal transport of foliarly-applied diclofop-methyl, shoot treatment was ineffective in inducing abnormal root morphogenesis of the seminal and lateral root systems, although it caused abnormalities of the coronal root system. Time course studies were initiated to examine the effect of root-applied diclofop-methyl at 0.05 μ M . Seminal root growth was inhibited (by diclofop-methyl) soon after treatment, while controls continued elongating. The distance between the seminal root apex and the first lateral primordia increased in the controls within one day after treatment, but decreased in the herbicide-treated roots. The distance between the seminal root apex and the first emerged lateral root was reduced by three days after treatment. The number of lateral primordia and emerged roots was unaffected three days after treatment. These dose-response and kinetic results suggested that diclofop-methyl caused a loss of apical dominance in the seminal root.