Effect of nickel at high concentration on proliferation of quiescent center cells and initiation of lateral root primordia in wheat seedlings

DNA synthesis and cell divisions in the quiescent center as well as initiation of lateral root primordia were investigated in the course of incubation of the roots of 3-day-old wheat (Triticum aestivum L.) seedlings on the medium with 0.1 mM NiSO₄ for 72 h. It was found that the earliest effect of nickel on proliferation of the quiescent center cells was associated with an increase in the mitotic index 6 h after the beginning of its action. This effect was assumed to depend on an increase in mitosis time. Twelve hours after the beginning of the effect of nickel, mitotic index became somewhat lower, and in 18 h it sharply decreased. Some dividing cells were observed among the initial cells of certain tissues and near the quiescent center even in 72 h. The portion of DNA synthesizing cell sharply decreased in 12 h, and in 48 h such cells were lacking. The main mechanism governing the termination of cell proliferation in the quiescent center as well as in the meristem and calyptrogen of the cap is the inhibition of cell transition to DNA synthesis. The cells that had time to start DNA synthesis or already finished it and were in other phases of the cycle continued a slow progression through the cycle and completed it. Sister cells, produced as a result of divisions, left the mitotic cycle in the phase G₁ and transited to dormancy. Nickel did not inhibit initiation and development of lateral root primordia. Resumption of DNA synthesis and cell divisions occurred not only in the pericycle and endodermis participating in the initiation of lateral root primordia but also in the cortex cells in the vicinity of developing primordia. In 18 h after the beginning of the experiment when the rate of the root growth considerably decreased, the region, where primordia were initiated, was located closer to the root tip. Subsequently, when elongation of the cells was inhibited, this region moved closer to the tip until structural disturbances occurred in the nuclei of the endodermal cells located near the root tip and elongated under the effect of nickel. The results concerning the effect of nickel and other heavy metals on root cell proliferation obtained by other researchers and the role of pericycle organization in the translocation and accumulation of nickel in the tissues are discussed.     

The timing of synthesis of proteins required for mitotic spindle and phragmoplast in partially synchronized root meristems of Vicia faba L

After cycloheximide treatment (1 h, 2.5 micrograms/ml) protein synthesis was decreased by 70% and was partially restored after 7 h of postincubation (still 20% decrease). In partially synchronized root meristems of Vicia faba L. treated with cycloheximide at middle G2, a strong decrease of the mitotic index was observed. Exposure to the drug at late G2 did not modify the mitotic index; the changes in the phase indices suggested that the course of mitosis was blocked at prophase-metaphase/anaphase-telophase transitions. The use of indirect immunocytochemical staining of tubulin (second antibody labeled with peroxidase) made it possible to show a decreased number of cells with preprophase bands in cycloheximide-treated meristems and the mitotic spindles and phragmoplasts containing a reduced number of shortened bands of microtubules. As a result of these structural and functional disturbances, binucleate cells and polyploid nuclei were observed.     

THE EARLY ACTION OF THE FLORAL STIMULUS ON MITOTIC ACTIVITY AND DNA SYNTHESIS IN THE APICAL MERISTEM OF XANTHIUM STRUMARIUM

Vegetative plants of Xanthium strumarium (a short-day species) were induced to flower by exposure to a single 16-hr long night. By cutting off the induced leaf (half-expanded leaf) at various times, it was established that, by 8 hr after the end of the long night, a sufficient amount of floral stimulus had reached the meristem to induce a flowering response. The following sequence of events occurred in both the peripheral and central zones of the apical meristem of induced plants: 1) a rise in the mitotic index beginning at 28 hr after the end of the long night and culminating at 36 and 56 hr; 2) a stimulation of DNA synthesis starting at 32–36 hr and reaching a maximum at 60 hr; 3) an increase in nucleolus diameter starting at 32 hr. The cell population in the meristems of both vegetative and induced plants displayed a similar distribution, with about 80 % of the nuclei with the 2C amount of DNA. The comparison of the kinetic data concerning the mitotic index and DNA synthesis indicated that one of the early effects of the floral stimulus in the peripheral and central zones was the release in mitosis of cells whose nuclei were in the postsynthetic (G₂) phase of the mitotic cycle. In the pith-rib meristem, the following events were recorded: 1) a stimulation of DNA synthesis starting at 20 hr; 2) a rise of the mitotic index beginning at 28 hr; 3) the vacuolation and elongation of cells starting at 48 hr. All these events occurred well before the initiation of bract and flower primordia, which began at 96 and 136 hr, respectively. Neither stimulation of mitotic activity nor flowering occurred in the meristems of plants subjected to a long night interrupted at its midpoint by a 5-min light break. The results are discussed in relation to the early events which are known to occur in the meristems of other photoperiodic species in transition to flowering.     

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.     

Heterogeneity in G2 duration during lateral root development

Developing lateral roots of V. faba were treated with 5-aminouracil for up to 6 hours using the 5-AU inhibition method discussed in this paper; the duration of G₂+mitosis/2 and the percentages of slow dividing cells were estimated from the fall in MI observed in just emerged meristems, very large primordia and large primordia. The results indicate that during the period of development studied here there are two subpopulations of dividing cells: 1) fast dividing population which makes up about 84 % of the dividing cells and which has a G₂+mitosis/2 duration of about 3.3 hours, and 2) a slow dividing population which constitutes about 16 % of the dividing cells and which has a G₂ duration in excess of 12 hours. This heterogeneity is discussed in relationship to the behaviour of different populations of proliferating cells during root morphogenesis.     

The effect of butyrate on the cell cycle in root meristems of Pisum sativum

Sodium butyrate at 5 mM in aerated White's medium reduced the mitotic index in root meristems of seedlings of Pisum sativum to < 1% after 12 h. This effect was lessened as the butyrate concentrations were lowered. The fraction of the root meristem nuclei in G2 increased to ~ 70% after 12 h in butyrate. After 12 h exposure to butyrate, seedlings transferred lo medium without butyrate gradually re-established their normal root meristem mitotic pattern, with a burst of mitosis at 10 h after the transfer. Even a brief exposure to butyrate inhibited DNA synthesis, and nuclei released from butyrate exposure were still unable to resume normal DNA synthesis even after 12 h. This information suggests that butyrate halts progression through the cell cycle by arresting meristem nuclei in G2 and inhibiting DNA synthesis.     

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.     

Hepatic proliferation after partial liver irradiation in Sprague–Dawley rats

The liver has powerful capability to proliferate in response to various injuries, but little is known as to liver proliferation after irradiation (IR) injury. This study investigated whether liver proliferation could be stimulated in low-dose irradiated liver by partial liver IR injury with high dose radiation. Sprague–Dawley rats were irradiated by 6-MV X-ray with single dose of 25 Gy to the right-half liver, while the left-half liver was shielded (0.7 Gy) or irradiated with single doses of 3.2, 5.6, and 8.0 Gy, respectively. Hepatic proliferation in the shielded and low-dose irradiated left-half liver was evaluated by serum hepatic growth factor (HGF), proliferating cell nuclei antigen (PCNA), liver proliferation index (PI), hepatocyte mitosis index (MI). The observation time was 0 day (before IR), 30, 60, 90, and 120 days after IR. Our results showed that serum HGF and hepatocyte HGF mRNA increased after IR with HGF mRNA peak on day 30 in the shielded and low-dose irradiated left-half livers, and their values increased as the dose increased to the left-half liver. Liver PI and PCNA mRNA peaked on day 60 with stronger expressions in higher doses-irradiated livers. MI increased after IR, with the peak noted on day 60 in the shielded and on day 90 in the low-dose irradiated left-half livers. There was a 30 day delay between MI peaks in the shielded and low-dose irradiated livers. In conclusion, 25 Gy partial liver IR injury could stimulate the shielded liver and low-dose irradiated liver to proliferate. In the livers receiving a dose range of 3.2–8.0 Gy, the proliferation was stronger in higher doses-irradiated liver than the low-dose irradiated. However, IR delayed hepatocyte mitosis.     

Effect of OA-inhibitor of protein phosphatases PP1 and PP2A — on initiation of DNA replication and mitosis in <Emphasis Type="Italic">Vicia faba</Emphasis> root meristems

According to the principal control point (PCP) hypothesis, experiments with excised, carbohydrate-starved stationary root meristems of Vicia faba var. minor have demonstrated that cells which previously divided asynchronously were preferentially blocked in G₁ (PCP1) and G₂ (PCP2) phases. When stationary phase meristems are supplied with exogenous carbohydrate (2 % sucrose), the G1- and G2-arrested cells start out DNA replication and mitotic divisions, respectively. The resumption of DNA synthesis and mitosis is not immediate and the delays of G₁- and G₂-arrested cells are found different. Using this model, we examined the effects of 4 pulse incubations with okadaic acid (OA), a specific inhibitor of PP1 and PP2A, on the duration of intervals elapsing between the provision of sucrose and the first appearance of S- and M-phase cells. We have found that depending on the period during which OA had been applied, the release from G1 and G2 phase arrest-points becomes prolonged, showing different time-course modifications. The obtained data provide evidence that activation of PP1 and PP2A is required to allow the cells for both PCP1→S and PCP2→M transitions in root meristems of V. faba.     

Effect of gamma irradiation on nucleolar activity in root tip cells of tetraploid Triticum turgidum ssp. durum L

Ionizing irradiation induces positive or negative changes in plant growth (M1) depending on the amount of irradiation applied to seeds or plant parts. The effect of 50–350 Gy gamma irradiation of kernels on nucleolar activity, as an indicator of metabolic activity, in root tip cells of tetraploid wheat Triticum turgidum ssp. durum L. cv. Orania (AABB) was investigated. The number of nucleoli present in nuclei and micronuclei as well as the mitotic index in the different irradiation dosages was used as an indicator of the cells entering mitosis, the chromosomes with nucleolar organizer regions that are active as well as chromosome doubling in the event of unsuccessful mitotic division. Nucleolar activity was investigated from 17.5 to 47.5 h after the onset of imbibition to study the first mitotic division and its consequences on the cells that were in G₂ and G₁ phases at the time of gamma irradiation. Untreated material produced a maximum of four nucleoli formed by the nucleolar organizing regions (NORs) on chromosomes 1B and 6B. In irradiated material, additional nucleoli were noted that are due to the activation of the NORs on chromosome 1A in micronuclei. The onset of mitosis was highly significantly retarded in comparison to the control due to checkpoints in the G₂ phase for the repairing of damaged DNA. This study is the first to report on the appearance of nucleoli in micronuclei as well as activation of NORs in the micronuclei that are inactive in the nucleus and the effect of chromosome doubling on nucleolar activity in the event of unsuccessful mitotic division.

     

CYTOKININ- AND GIBBERELLIC ACID-INDUCED EFFECTS ON THE STRUCTURE AND METABOLISM OF SHOOT APICAL MERISTEMS IN OPUNTIA POLYACANTHA (CACTACEAE)

The dormant axillary buds of Opuntia polyacantha can be activated by either cytokinins or gibberellic acid. Under the influence of benzylaminopurine (BAP), the axillary bud meristem increases greatly in size and becomes mitotically active. The primordia produced by the meristem develop as normal photosynthetic leaves. Gibberellic acid (GA) also causes the meristem to become mitotically active, but the meristem does not increase in size. The primordia produced under the influence of GA develop as normal cactus spines. Leaf-producing meristems and spine-producing meristems have the same zonation, despite the differences in size. The meristems are composed of a uniseriate tunica, a central mother cell zone, peripheral zone, and a pith rib meristem. The mitotic activity of each of the zones in the leaf-producing meristem differs significantly from the mitotic activity of the corresponding zones in the spine-producing meristem.     

Proliferation kinetics of mouse tongue epithelium under normal conditions and following single dose irradiation

Epithelial proliferation in the ventral surface of mouse tongue follows a pronounced circadian rhythm with a peak in mitotic activity at 10.00 a.m., preceded by a wave of DNA synthesis 8 h earlier. Nearly all cells (85%) pass through G2 and mitosis immediately after the S-phase; they subsequently divide again, usually after 2 or 3 days, indicating cohorts of cells with different G1-duration. The fraction of all nucleated cells comprised in one daily proliferation wave is about 20%, indicating a turnover time of the nucleated cell compartment of about 5 days. Cytotoxic injury by a single radiation dose of 20 Gy causes a steep decrease in cell counts, leading to complete denudation after 9–13 days. The difference between the latent period before ulceration and the tissue turnover time is explained by a marked proliferative activity of the doomed cells. The mitotic index increases steeply after day 1 to three times the control level, but most mitotic figures display gross abnormalities such as multipolar spindles or chromosome clumping. As a consequence cells with abnormal or multiple nuclei appear in the basal layers 3 days post irradiation and subsequently migrate to the upper layers. After denudation the epithelium rapidly becomes restored, with a phase of transient hyperplasia on days 13–14. Normal architecture is regained by day 15. Over the whole healing period the mitotic index remains at a high level, with most of the mitoses appearing histologically normal.     

Imbalance between apoptosis and proliferation causes late radiation damage of salivary gland in mouse

Severe xerostomia is a common late radiation consequence, which occurs after irradiation of head and neck malignancies. The aim of the present study was to analyze apoptosis and proliferation and their relationship during the late post-irradiation phase. C57BL/6 mice were locally irradiated in head and neck region with a single dose of 7.5 or 15 Gy and their submandibular glands were collected at 40 and 90 days after irradiation. To identify apoptotic cells, the TUNEL method was employed and immunohistochemistry with proliferating cell nuclear antigen (PCNA) was used for detecting proliferation. Histological changes at day 40 were mild in contrast to day 90 when glands of irradiated mice showed severe atrophy, vacuolization and mononuclear infiltration. Acinar cells, granular and intercalated duct cells of mice irradiated with 7.5 and 15 Gy expressed higher apoptotic index than cells of non-irradiated, control glands at both examined time points. At 40 days, a higher proliferation index in granular and intercalated duct cells was detected only in group irradiated with 7.5 Gy. At 90 days, proliferation index for all cell types in both irradiated groups was similar to the controls. According to our results, the imbalance between apoptosis and proliferation caused by X-irradiation may be the reason for gland impairment during the late post-irradiation phase.     

Proliferation kinetics of mouse tongue epithelium under normal conditions and following single dose irradiation.

Epithelial proliferation in the ventral surface of mouse tongue follows a pronounced circadian rhythm with a peak in mitotic activity at 10.00 a.m., preceded by a wave of DNA synthesis 8 h earlier. Nearly all cells (85%) pass through G2 and mitosis immediately after the S-phase; they subsequently divide again, usually after 2 or 3 days, indicating cohorts of cells with different G1-duration. The fraction of all nucleated cells comprised in one daily proliferation wave is about 20%, indicating a turnover time of the nucleated cell compartment of about 5 days. Cytotoxic injury by a single radiation dose of 20 Gy causes a steep decrease in cell counts, leading to complete denudation after 9-13 days. The difference between the latent period before ulceration and the tissue turnover time is explained by a marked proliferative activity of the doomed cells. The mitotic index increases steeply after day 1 to three times the control level, but most mitotic figures display gross abnormalities such as multipolar spindles or chromosome clumping. As a consequence cells with abnormal or multiple nuclei appear in the basal layers 3 days post irradiation and subsequently migrate to the upper layers. After denudation the epithelium rapidly becomes restored, with a phase of transient hyperplasia on days 13-14. Normal architecture is regained by day 15. Over the whole healing period the mitotic index remains at a high level, with most of the mitoses appearing histologically normal.     

Autoradiographic Examination of Meristems of Intact Boron-deficient Squash Roots Treated with Tritiated Thymidine

Intact roots of boron-sufficient squash (Cucurbita pepo L.) plants, plants entering boron deficiency, and plants recovering from boron deficiency were exposed to tritiated thymidine at the end of the treatment period to label the replicating DNA of root tip cells. Using histological sections, autoradiographs of intact root meristems were prepared. The labeling pattern in +B root tips revealed the presence of a well defined quiescent center. The ability of root tip cells to incorporate label is correlated with the total root elongation during the −B treatment period. A greater amount of total root elongation during boron deficiency and recovery reflects the fact that root tip cells have retained their ability to synthesize DNA and enter mitosis for a longer time. In roots recovering from boron deficiency, cells of the quiescent center were seen to play no part in the recovery process in roots treated for as long as 20 hours in a −B nutrient solution. They were inactive before, during, and after the −B treatment. Cessation of mitosis occurs as early as 6.5 hours after boron is withheld from the nutrient solution while DNA synthesis can occur for as long as 20 hours after withholding boron. It was concluded that boron is essential for continued DNA synthesis and mitotic activity. The absence of boron results in the cessation of mitosis and DNA synthesis within 20 hours from the time boron is withheld.     

Enhancement of transformation rates in higher plants by low-dose irradiation: Are DNA repair systems involved in the incorporation of exogenous DNA into the plant genome?

Irradiation (X-ray; 5–15 Gy) of protoplasts treated with plasmid-DNA and PEG yielded higher transformation rates in comparison to non-irradiated protoplasts transformed by the same method. This could be demonstrated for four plant species. The irradiation doses used did not affect the total number of colonies regenerated without selection pressure, but resulted in 3–6-fold enhancement of hygromycin- or kanamycin-resistant colonies. Plant regeneration frequencies of transformed colonies derived from irradiated and non-irradiated protoplasts were similar in tobacco as well as in Petunia. Higher integration rates of foreign DNA as a consequence of an increased recombination machinery in irradiated cells may be responsible for the enhancement of the number of stably transformed colonies.     

DNA synthesis and mitotic activity in cells of regenerating rat liver following x-irradiation in stages GO and Gl]

Effect of X-rays on DNA synthesis and mitotic activity of regenerating liver cells has been studied. The irradiation was performed at a dose of 630 rad before hepatectomy and 2.5 and 6 hours after the stimulation of liver. With the stimulated liver being irradiated, the number of cells synthetizing DNA and entering into mitosis was seen reduced almost twice, whereas DNA synthesis and entering into mitosis were delayed, resp., by 4 and 6 hours. Irradiation of liver before the stimulation brings about a delay in DNA synthesis and in start of mitosis by 2 and 4 hours, resp., without reducing the numbers of cells capable to synthesize DNA and to enter mitosis.     

Transmission of genome damage from irradiated male rats to their progeny

The effect of gamma-radiation (3Gy) on slowly proliferating liver tissue of male rats and their progeny was investigated with respect to induction and duration of latent damage. The irradiation caused latent cytogenetic damage in the liver in irradiated males of the F(0) generation, which manifested itself in different ways during proliferation of hepatocytes induced by partial hepatectomy: a reduced proliferating activity, a higher frequency of chromosomal aberrations and a higher proportion of cells with apoptotic DNA fragments were observed, compared with non-irradiated rats. In the progeny of irradiated males (F(1) and F(2) generation), the latent genome damage manifested itself during regeneration of the liver after partial hepatectomy by similar, but less pronounced changes compared with those seen in irradiated males of the parental generation. This finding gave evidence of the transfer of part of the radiation-induced genome damage from parents to their offspring. Irradiation of F(1) and F(2) progeny of irradiated males (their total radiation load being 3 + 3 and 3 + 0 + 3 Gy, respectively) caused less change as irradiation of progeny of non-irradiated control males (their total radiation load being 0 + 3 and 0 + 0 + 3 Gy, respectively).