Changes in mitotic indices in roots of Vicia faba

Roots of Vicia faba were treated with solutions of colchicine or IAA or both. Mitotic indices and the frequencies of the different stages of mitosis were determined immediately after a three hour treatment or following a 24 hour period of recovery. Roots scored after treatment with colchicine for three hours showed several effects, none of which were reversed by simultaneous treatment with IAA. Treatment with IAA for three hours had little detectable effect on mitotic index (MI) on the frequencies of the various stages of mitosis. After a recovery period, following a three hour treatment, of 24 hours, colchicine treated roots showed a significant increase in their MI; this was due largely to an increase in the number of metaphases but it was also due in part to the presence of tetraploid cells in division. IAA treated roots revealed an inhibition of mitotic activity, which was most marked at 3.13–6.26×10^–4 M IAA. The results from roots treated with mixtures of colchicine and IAA for three hours and fixed 24 hours later showed: 1) the increase in MI induced by colchicine is reversed by IAA, the intensity of the reversal increasing with increasing concentrations of IAA; 2) reductions in the total numbers of cells in prophase or in metaphase occur after treatment with different concentrations of IAA; 3) IAA leads to a reduction in the number of tetraploid cells seen in division.It appears that colchicine induces a change in the pattern of mitotic activity 24 hours after the end of treatment and its effects are reversed by IAA. At 4.2×10^–4 M IAA a balance occurs between the opposing effects of colchicine and IAA and the MI is not significantly different from that of the controls. It is suggested that one result of a treatment with colchicine is a change in the level of growth factors in root meristems. This change, which appears to result in a temporary increase in MI is reversed by the addition of IAA. Thus one of the growth factors, the level of which has been affected, is replaceable by exogenous IAA.     

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.     

A Differential Response to Colchicine of Meristems of Roots of Vicia faba

In whole root systems of Vicia faba, including primaries andlaterals, the meristems do not respond in a uniform manner totreatment with colchicine. The meristems of the primary andfully emerged lateral roots become mixoploid and these rootsshow a temporary inhibition of growth. Lateral roots that emergewithin 48 hours of treatment show no effects of colchicine;their growth is not inhibited and they contain few or no polyploidcells. The cells of the primordia that produce these lateralsappear to be insensitive to colchicine: at the insensitive stagethese primordia contain at least 1, 000 cells. Primordia withless than 800 cells are very sensitive to colchicine. They donot recover from treatment and appear to be completely inhibited.The stages of extreme sensitivity and resistance are transientphases in the morphogenesis of a lateral root. The change insensitivity to colchicine is accompanied by a fall in the mitoticindex; this is highest in young primordia, where it is abouttwice the value found in growing laterals.     

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.     

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.     

Regeneration mechanisms of ontogenetic radioadaptation in plants

Experimental data were obtained that in pea seedlings modified by decapitation of main root had increased radioresistance (radioadaptation), fixed by various parameters of growth activity of lateral roots, and decreased ability to repair sublethal damages, detected by method of acute gamma-irradiation dose fractionation. These facts both with enlargement of dose dependence shoulder in lateral roots of decapitated seedlings led to conclusion that main role in such mechanism of radioadaptation effect of decapitation belongs to supercellular processes such as repopulation and regeneration. Conclusion was confirmed by the additional comparative investigations of cyto- and histological parameters of apical meristems of intact (control) and decapitated (experiment) lateral roots. It was shown, that the decapitated seedlings had increased mitotic activity of apical meristems of lateral roots and total volume of their meristematic zone. So at the moment of application of irradiation in the test-dose decapitated variant had significantly more meristematic cells of certain size that allowed biological object to form necessary (critical) amount of elements for valid or more complete postradiation recovery.     

Effects of cadmium on meristem activity and nucleus ploidy in roots of Pisum sativum L. cv. Frisson seedlings

The effects of cadmium (Cd) administration on primary root growth, mitotic activity of apical meristems, mitotic aberrations and percentage of nucleus ploidy classes of differentiated roots were examined in Pisum sativum L. cv. Frisson. Cadmium caused a reduction of root length related to concentration, with an almost complete block of growth in plants treated with 250 μM Cd, from 24 h of treatment. Root lengthening is generally related to apical meristem activity, however, in the examined pea plants, mitotic activity was suppressed by 2.5 and 25 μM Cd treatment, while the highest Cd concentration, 250 μM, caused the occurrence of mitotic figures consisting almost exclusively of prophases. The lack of relation between root lengthening and mitotic activity was explained by the meristematic activity in the first period of treatment and by a different cell elongation. Lower (0.25, 0.5 and 1 μM), non-blocking Cd concentrations induced a number of mitotic aberrations, mainly consisting of sticky metaphases and anaphase bridges, whose frequency increased with Cd concentration. Besides, Cd induced variations of the percentages of nucleus populations in the differentiated roots, increasing the percentage of 4C nuclei and decreasing that of 2C. The mechanisms involved in the nuclear response to Cd, and the possible relations between Cd alteration of meristem cell activity and nuclear ploidy of differentiated cells are discussed.     

The Emergence and Early Growth of the Lateral Root in Vicia faba L.

The duration of the mitotic cycle, as well as the proportionof cells with long and short cycle times and quiescent cells,have been investigated in the apical meristems of young lateralroots of Vicia faba. No changes took place in the duration ofC or in the phases of the mitotic cycle as the lateral rootemerged from the primary root, though the proportion of proliferatingcells increased and the quiescent fraction of cells decreased.It is suggested that the low frequency with which newly emergedlateral roots label with ³H-TdR is a result of the formationof a large endogenous pool of TdR in the meristems during theperiod they are temporarily quiescent. The changes which tookplace in the parameters of cell proliferation during the earlygrowth of the lateral root have been correlated with those inroot apical meristems following the onset of seed germination.     

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.     

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.     

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.     

Possibility of synchronizing cell populations in embryonic barley meristem

Cell populations of the apical root parts, stem embryo and the leaf of barley seedlings are found to have different sensitivity to the synchronizing effect of 5-aminouracil, low temperature (+2 degrees C) and colchicine. The effect of 5-aminouracil and low temperature in the presence of colchicine proved to be the most effective in respect to synchronization of the root meristem cell populations. It also increases significantly the mitotic activity in the stem embryo and leaf meristems. The leaf meristem is more sensitive to low temperature as compared to the stem embryo meristem.     

Differential spatial expression of A- and B-type CDKs, and distribution of auxins and cytokinins in the open transverse root apical meristem of Cucurbita maxima

Background and Aims

Aside from those on Arabidopsis, very few studies have focused on spatial expression of cyclin-dependent kinases (CDKs) in root apical meristems (RAMs), and, indeed, none has been undertaken for open meristems. The extent of interfacing between cell cycle genes and plant growth regulators is also an increasingly important issue in plant cell cycle studies. Here spatial expression/localization of an A-type and B-type CDK, auxin and cytokinins are reported in relation to the hitherto unexplored anatomy of RAMs of Cucurbita maxima.

Methods

Median longitudinal sections were cut from 1-cm-long primary root tips of C. maxima. Full-length A-type CDKs and a B-type CDK were cloned from C. maxima using degenerate primers, probes of which were localized on sections of RAMs using in situ hybridization. Isopentenyladenine (iPA), trans-zeatin (t-Z) and indole-3yl-acetic acid (IAA) were identified on sections by immunolocalization.

Key Results

The C. cucurbita RAM conformed to an open transverse (OT) meristem typified by an absence of a clear boundary between the eumeristem and root cap columella, but with a distinctive longitudinally thickened epidermis. Cucma;CDKA;1 expression was detected strongly in the longitudinally thickened epidermis, a tissue with mitotic competence that contributes cells radially to the root cap of OT meristems. Cucma;CDKB2 was expressed mainly in proliferative regions of the RAM and in lateral root primordia. iPA and t-Z were mainly distributed in differentiated cells whilst IAA was distributed more uniformly in all tissues of the RAM.

Conclusions

Cucma;CDKA;1 was expressed most strongly in cells that have proliferative competence whereas Cucma;CDKB2 was confined mainly to mitotic cells. iPA and t-Z marked differentiated cells in the RAM, consistent with the known effect of cytokinins in promoting differentiation in root systems. iPA/t-Z were distributed in a converse pattern to Cucma;CDKB2 expression whereas IAA was detected in most cells in the RAM regardless of their proliferative potential.     

PRIMORDIAL LEAF AND PHYTOHORMONE EFFECTS ON EXCISED SHOOT APICAL MERISTEMS OF COLEUS BLUMEI BENTH.

Coleus blumei Benth. apical meristems and apical meristems +1, +2, +3 primordial leaf pairs were cultured to examine phytohormone influences on development and correlative effects of developing primordial leaves on in vitro responses. The meristem with no phytohormones or low levels of IAA could not develop in vitro. At least 0.1 mg/l IAA and optimumly 1-2 mg/l IAA were required for development into complete plants. IAA from 0.1 to 3 mg/l also resulted in root development with no apparent leaf or shoot formation. Levels of IAA higher than 3 mg/l were inhibitory to development. Kinetin, as a substitute for naturally occurring cytokinins, alone (0.0003 to 3 mg/l) resulted in development of rosettes of leaves. In the presence of IAA (***1 mg/l) and kinetin (0.003 mg/l) plants, rosettes, individual leaves with roots, and roots developed from isolated meristems. Glutamine and adenine sulfate both appeared inhibitory to meristem development. With +1, +2, +3 developing primordial leaf pairs left attached to the apical dome, three pairs were required for plant formation in the absence of phytohormones. In the presence of IAA, two pairs of primordial leaves resulted in plant formation; whereas, with IAA and low levels of kinetin one pair of primordial leaves was enough. Higher levels of kinetin were inhibitory to plant development with primordial leaves present. ABA appeared to be inhibitory to development of meristems and meristems +1, +3 primordial leaves at low concentrations and resulted in death at ***1 mg/l. Developing primordial leaves appear to supply the apical meristem with a balance of phytohormones during growth. Meristem development into a plant first involved formation of leaf primordia. Establishment of a bipolar axis with root formation followed.     

Synergistic interaction between coumarin 1,2-benzopyrone and indole-3-butyric acid in stimulating adventitious root formation in Vigna radiata (L.) Wilczek cuttings: I. Endogenous free and conjugated IAA and basic isoperoxidases

Cuttings from 7-day-old Vigna radiata seedlings were treated for 24 h with various concentrations of coumarin and/or indole-3-butyric acid (IBA), applied either alone or in combination, in order to stimulate adventitious root formation (ARF). The effects of treatment on endogenous free and conjugated indole-3-acetic acid (IAA), basic peroxidase (basic PER) activity and its isoperoxidases analysis and their relation to ARF were then investigated at the potential rooting sites during the first 96 h after application. Simultaneously, combined treatments acted synergistically in inducing more adventitious roots in treated cuttings than in those treated with coumarin or IBA individually, as compared with the control. Endogenous free IAA increased transiently in treated cuttings as compared with the control and the maximum increase occurred with the combined treatment. This suggests that coumarin and IBA may act synergistically in increasing the endogenous free IAA level during the induction phase of rooting to initiate more roots. Likewise, higher level of conjugated IAA was also found in treated cuttings than in untreated ones, during the primary events of ARF, with the maximum level occurring in the combined treatment. Comparison of the dynamics of conjugated IAA and activity of basic PERs led to conclusion that the former but not the latter is responsible for downregulation of endogenous IAA levels significantly during the primary events of ARF. A sharp increases in basic PERs occurred during the secondary events of ARF, suggesting their role in root initiation and development rather than root induction.     

Water hyacinth (Eichhornia crassipes) to biomonitor genotoxicity of low levels of mercury in aquatic environment

The mitotic cell-cycle duration of root meristematic cells of Eichhornia crassipes as determined by the colchicine labelling method was approximately 24 h at 30 +/- 1 degrees C. In one experiment the intact root meristems of E. crassipes were subjected to 1 h acute exposure to water contaminated with maleic hydrazide (MH), 56 ppm, or methyl mercuric chloride (MMCl), 0.1-0.5 ppm, followed by recovery in tap water for 4-48 h. In a second experiment the roots were subjected to 96 h exposure to water contaminated with MH, 56 ppm, or MMCl, 0.0001-0.1 ppm. In both experiments the cytological end-point measured was the frequency of cells with micronuclei (MNC). In the first experiment, while in the MH-exposed root meristems the frequency of MNC was significant at 40 h of recovery, MMCl induced significant MNC at 12, 20, 24, 40, and 40 h of recovery depending on the concentration. In the second experiment both test chemicals induced MNC which was concentration-dependent in case of MMCl. The highest ineffective concentration tested (HICT) and lowest effective concentration tested (LECT) for MMC determined in this experiment were 0.0005 ppm and 0.001 ppm, respectively. The present work provides evidence that E. crassipes could be a promising in situ environmental biomonitoring assay system.     

Roles for Class III HD-Zip and KANADI genes in Arabidopsis root development

Meristems within the plant body differ in their structure and the patterns and identities of organs they produce. Despite these differences, it is becoming apparent that shoot and root apical and vascular meristems share significant gene expression patterns. Class III HD-Zip genes are required for the formation of a functional shoot apical meristem. In addition, Class III HD-Zip and KANADI genes function in patterning lateral organs and vascular bundles produced from the shoot apical and vascular meristems, respectively. We utilize both gain- and loss-of-function mutants and gene expression patterns to analyze the function of Class III HD-Zip and KANADI genes in Arabidopsis roots. Here we show that both Class III HD-Zip and KANADI genes play roles in the ontogeny of lateral roots and suggest that Class III HD-Zip gene activity is required for meristematic activity in the pericycle analogous to its requirement in the shoot apical meristem.     

QUANTITATIVE STUDIES OF THE ROOT APICAL MERISTEM OF EQUISETUM SCIRPOIDES

An investigation was made of the meristematic activity of the apical cell, its immediate derivatives (merophytes), and of other selected cell populations of the root of Equisetum scirpoides Michx. The plane of the first division of a derivative of the apical cell is radiallongitudinal, which provides evidence that merophytes immediately adjacent to the apical cell cannot be the ultimate root initials. The apical cell is as active mitotically in roots 20–40 mm long as it is in roots that are 0.25–1 mm in length. The mitotic activity of the apical cell and of other cell populations was determined from the mitotic index, and from determination of the durations of the cell cycle and of mitosis of the apical cell by using the colchicine method of metaphase accumulation. Microspectrophotometric measurements of DNA content indicated that there was no consistent increase in DNA (endopolyploidy) in the apical cell or in the other meristematic cells as roots increased in length. Conclusion: there is no evidence that the apical cell becomes quiescent or undergoes endopolyploidy as a root increases in length.