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.     

DURATION OF CELL CYCLES IN CULTURED ROOTS OF CONVOLVULUS

The durations of the cell cycle in physiologically different regions of the meristem of cultured roots of Convolvulus arvensis were determined by the metaphase-accumulation technique involving colchicine. The cell cycle in the root cap increases from 13 hr in the actively dividing initials of the first tier to 155 hr in the slowly dividing initials of tiers 2–4 to an indeterminate value for derivatives of the initials in the root cap columella. The cycle times for the cells of the central cylinder and cortex are 21 and 27 hr, respectively. The cells of the quiescent center have a cycle of an estimated 420 hr. The duration of the cell cycle in these different regions is discussed in relation to the increased duration of G₁ in slowly or non-dividing cells. The possible regulation of cell division by the synthesis of a cell-division factor in the quiescent center is also discussed.     

MITOTIC ACTIVITY IN THE ROOT APICAL MERISTEM OF AZOLLA FILICULOIDES LAM., WITH SPECIAL REFERENCE TO THE APICAL CELL

The meristematic activity of the apical cell and its derivatives (merophytes) in the unbranched, determinate roots of Azolla filiculoides Lam. was investigated. The plane of division of the apical cell indicates that it is the initial of each merophyte. The division plane of each newly formed merophyte is strictly periclinal to the root surface and provides confirmation that the immediate derivatives of the apical cell cannot be the ultimate root initials. The frequency of cell division as determined by the mitotic index, and by the duration of the cell cycle as determined by the colchicine method, confirmed the meristematic activity of the apical cell. As roots increase in length, the duration of the cell cycle in the total meristem increases, with the apical cell possessing the longest cell cycle, whereas the immediate derivatives maintain approximately the same cycle duration as in shorter roots. In determinate Azolla roots, cell division appears to play a major role up to a certain root length, then increase in length is produced mainly by cell elongation.     

MITOTIC ACTIVITY IN THE ROOT APEX OF THE WATER FERN MARSILEA VESTITA HOOK. AND GREV.

Roots of Marsilea vestita ranging from 1–120 mm in length, as well as root primordia, were analyzed to determine mitotic activity and ploidy levels in the apical cell, five well-defined regions of the root proper, and two regions in the root cap. The mitotic index of the apical cell tended to be above the overall mean mitotic index for the entire apical meristem. No diurnal rhythm in mitotic index was apparent. The cell-cycle duration of the apical cell ranged from 12.1–25.2 hr, that of other regions of the root from 16.1–41.5 hr. There was no indication of polyploidy in any part of the apical meristem except in a few procambial cells. Thus, the results support the classical concept that the apical cell is the ultimate source of cells in the root.     

ZONATION IN THE APICAL CELL OF ZONARIA

Light and electron microscopic techniques were used to study the row of apical cells that form the meristem of the dictyotalean brown alga, Zonaria farlowii. A distinctly polar pattern occurs in the cells. Four ultrastructurally different cytoplasmic zones have been seen: an apical zone, a nuclear zone, a compound vacuolar zone, and a vegetative zone. The apical cell of Zonaria is a differentiating cytoplasmic unit where striking intracellular gradients occur.     

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.     

MITOTIC ACTIVITY AT THE SHOOT APEX OF AZOLLA FILICULOIDES

The mosquito fern, Azolla filiculoides Lam., was grown in a growth chamber on a nitrogen-free culture solution at 24 C under the following photoperiod: 16 hr light/8 hr darkness. Shoot tips were fixed every 2 hr for 24 hr to determine the mitotic index for the apical cell, immediate derivatives, and remaining cells to the level of the first leaf or lateral shoot primordium. Mitotic indices were 6.9%, 6.5% and 6.3%, respectively. The colchicine method was employed to determine the cell-cycle durations and duration of mitosis for the same populations of cells. The cell-cycle duration and duration of mitosis of the apical cell were 28.2 hr and 2.8 hr, respectively; for the immediate derivatives, 26.7 hr and 2.5 hr; for the remaining cells, 23.6 hr and 2.1 hr. Conclusions: the apical cell is as mitotically active as its immediate derivatives, and there is no evidence of a quiescent apical cell.     

CELL PROLIFERATION IN COMPLEX TISSUES: THE CONTROL OF THE MITOTIC CYCLE OF CELL POPULATIONS IN THE CULTURED ROOT MERISTEM OF SUNFLOWER (HELIANTHUS)

Experiments were performed with cultured primary root tips of sunflower (Helianthus annuus var. Russian Mammoth) to determine: (1) if progression in the mitotic cycle of meristematic cells was nutritionally controllable by carbohydrate starvation and replenishment; (2) where in the mitotic cycle control was effected; and (3) whether nutritional deprivation could be used to detect phenotypically different subpopulations in a complex tissue. Meristematic cells were rendered stationary by carbohydrate starvation, as indicated by the absence of cell division; this condition was reversed by carbohydrate provision. After 72 or 96 hr of starvation most cells stopped in G1 (80–90%) and G2 (10–20%), and a very few (“leaky” cells) continued to enter S. “Leaky” cells represent a small population with an S period of approximately 4.1 hr that either lack a principal control point in G1 or have an unusual metabolism whereby the control point requirements are met and have a carbohydrate dependence for mitosis. Though phenotypically different, no specific functions can be attributed to “leaky” cells at this time.     

EVIDENCE FROM THYMIDINE-3H-LABELED MERISTEMS OF VICIA FABA OF TWO CELL POPULATIONS

Treatments with tritiated thymidine (TdR-³H) have revealed the existence of two populations of mitotically active cells in meristems of lateral roots of Vicia faba. A rapidly dividing population, with a cycle time of 14 hr, constitutes about half the cells in the meristem. A second population of cells, with a cycle time in excess of 30 hr, is also present. Estimates of the relative size of this slowly dividing population are more difficult to make, but we calculate that this population includes 27–43% of meristem cells. The remaining fraction of the meristem is made up of cells that divide rarely or not at all. Since, at all times, both populations contribute to the mitotic index, the curve of the percentage of labeled mitoses that can be determined after a pulse label with TdR-³H differs from the curve expected of an ideal population in an important way: the peak value of the curve of the percentage of labeled mitoses is always less than 100%, usually between 75 and 80%. This heterogeneity within a meristem must be borne in mind in terms of the response of meristems to disruptive treatments, the mechanisms controlling mitotic cycle duration, and the spatial organization of a heterogeneous population in an organ that shows polarized growth.     

ONTOGENY OF THE INFLORESCENCE OF SAURURUS CERNUUS (SAURURACEAE)

The spicate inflorescence of Saururus cernuus L. (Saururaceae) results from the activity of an inflorescence apical meristem which produces 200–300 primordia in acropetal succession. The inflorescence apex arises by conversion of the terminal vegetative apex. During transition the apical meristem increases greatly in height and width and changes its cellular configuration from one of tunica-corpus to one of mantle (with two tunica layers) and core. Primordia are initiated by periclinal divisions in the subsurface layer. These are “common” primordia, each of which subsequently divides to produce a floral apex above and a bract primordium below. The bract later elongates so that the flower appears borne on the bract. All common primordia are formed by the time the inflorescence is about 4.4 mm long; the apical meristem ceases activity at this stage. As cessation approaches, cell divisions become rare in the apical meristem, and height and width of the meristem above the primordia diminish, as primordia continue to be initiated on the flanks. Cell differentiation proceeds acropetally into the apical meristem and reaches the summital tunica layers last of all. Solitary bracts are initiated just before apical cessation, but no imperfect or ebracteate flowers are produced in Saururus. The final event of meristem activity is hair formation by individual cells of the tunica at the summit, a feature not previously reported for apical meristems.     

DNA MICROSPECTROPHOTOMETRY OF SHOOT APICAL MERISTEM CELL POPULATIONS IN CERATOPTERIS THALICTROIDES (FILICALES)

A microspectrophotometric analysis of the DNA content of cell populations in the shoot apical meristem of young and adult stages of the filicalean fern Ceratopteris thalictroides was conducted to determine if the previously reported uptake of labeled DNA precursors by the apical cell was a consequence of endomitotic DNA replication or of DNA synthesis preceeding mitosis. Results demonstrate that in both the young and adult plants the estimated DNA content of the apical cell nuclei parallels that of the other cells of the meristem. There was no evidence of an “apical zone” of endopolyploid cells.     

Occurrence of two cell subpopulations with different cell-cycle durations in the central and peripheral zones of the vegetative shoot apex of Sinapis alba L.

The cell-cycle duration and the growth fraction were estimated in the vegetative shoot apical meristem of Sinapis alba L. The length of the cell cycle was about 86 h, i.e. 2.5 times shorter than the cell-doubling time (M. Bodson, 1975, Ann. Bot. 39, 547–554) and the growth fraction was between 32 to 41%. These data demonstrated that the cell population of this meristem was heterogeneous, including one subpopulation of rapidly cycling cells and one subpopulation of non-cycling cells, i.e. cells with a very long cell cycle compared with that of the rapidly cycling cells. Non-cycling cells had no particular localization within the meristem. Both the central and peripheral zones of the meristem were mosaics of rapidly cycling and non-cycling cells.Abbreviations G₁ pre-DNA-synthesis phase - G₂ post-DNA-synthesis phase - GF growth fraction - M mitosis phase - PLM pulse-labelled-mitoses method - S DNA-synthesis phase - T cell-cycle duration - TdR thymidine     

VARIATION IN CELL-CYCLE TIME AND NUCLEAR DNA CONTENT IN THE APICAL MERISTEM OF HELIANTHUS ANNUUS L. DURING THE TRANSITION TO FLOWERING

The mitotic cycle in the apical meristem of Helianthus annuus L. has been investigated during the transition to flowering. Towards the end of the strictly vegetative phase 8 days after sowing the average cell-cycle time, measured by colchicine-induced metaphase accumulation, was 37 hr in the peripheral zone, 83 hr in the central zone and 118 hr in the rib meristem. By Day 12 the cycle had shortened in all zones. By the time of floral initiation on Day 16 the cycle time had returned to its original value in the peripheral zone and the rib meristem, while in the central zone it continued to shorten to 33 hr, approaching the cycle time of the peripheral zone. Cytophotometric measurements of nuclear DNA showed that mitotic activation of the central zone was not associated with any reduction in the proportion of nuclei with a 4 C DNA content. It was calculated that the spatial and temporal variation in cell-cycle time was mainly a function of the length of the G₁/G₀ phase which lasted about 19 hr in the peripheral zone, 82 hr in the rib meristem, and declined from 55 to 21 hr in the central zone.     

THE EFFECT OF ESTRADIOL ON THE CELL KINETICS IN THE UTERINE AND CERVICAL EPITHELIUM OF NEONATAL MICE

The effect of estradiol-17β on the length of the various phases of the cell cycle was studied in the neonatal mouse uterine, and cervical epithelium. A double labelling method was used, and in addition labelled mitoses were counted. In the uterus proper, estradiol shortens the length of the total cell cycle, T_c, from 17-9 hr to 15-7 hr, and the duration of S phase, T_s, from 6–7 to 5-1 hr 6 hr after estradiol treatment. 12 hr after estradiol treatment, T_c is shortened to 7-4 hr and T_s to 4–5 hr. The shortening of T_c at 12 hr is mainly due to an effect on T_G1, which is shortened from 8–55 hr in untreated animals to 1–8 hr in estradiol treated animals. The T_c of cervix epithelium cells in untreated animals was found to be 21-8 hr. After treating the mice for 6 hr with estradiol the T_c was now increased to 47 hr and further to 61 -2 hr following 12 hr treatment with the hormone. T_s increases from 8-3 hr to 15-2 hr following 6 hr estradiol treatment, and to 15-4 hr after 12 hr treatment. The effect is most pronounced in T_G1, which is lengthened from 10–95 hr in untreated animals to 28-1 hr and 43 hr, respectively, in animals treated for either 6 or 12 hr with estradiol.     

BISACK ANALYSIS OF THE PHYTOHAEMAGGLUTININ-INDUCED PROLIFERATION OF HUMAN PERIPHERAL LYMPHOCYTES

The rate of stimulation as well as subsequent cell cycle duration was examined in phytohaemagglutinin-stimulated human peripheral lymphocytes grown in vitro in the presence of non-inhibitory concentrations of bromodeoxyuridine. After incorporation of this heavy atom analogue of thymidine into replicating cellular DNA, it was possible to identify unequivocally metaphase cells which had replicated for one, two and three or more cell cycles. Utilizing this technique, distribution curves were obtained for the appearance of metaphase cells in successive generations, were analysed by a computer simulation model, and the rate of stimulation (4.5% per hr of the remaining unstimulated population) and cell cycle duration (12·3 hr) were determined. The results were compared with those obtained by autoradiography and the possible relationship to the ‘transition probability’ model for cellular proliferation is discussed.     

SHOOT HISTOGENESIS: THE EARLY EFFECTS OF GIBBERELLIN UPON STEM ELONGATION IN TWO ROSETTE PLANTS

Sachs , Roy M., Charles F. Bretz , and Anton Lang . (U. California, Los Angeles.) Shoot histogenesis: The early effects of gibberellin upon stem elongation in two rosette plants. Amer. Jour. Bot. 46(5): 376–384. Illus. 1959.—Within 24 hr. after the application of gibberellic acid (GA) to vegetative plants of biennial Hyoscyamus and of the long-day plant Samolus, a considerable increase in mitotic activity was observed in the pith, cortical, and vascular tissues of the rosette axis immediately below the apical meristem. As the treatment continued, the zone of cell division increased commensurate with the increase in length of the stem; the new cell divisions formed transverse walls predominantly and thus contributed to stem elongation. The cell contribution from the apical meristem was but a small fraction of the total produced by the subapical tissues, suggesting that the induced subapical mitotic activity is the main site of tissue development in the shoot. There was no evidence for cell elongation for at least 72 hr. after application of GA, and, hence, the initial increase in stem length was due solely to an increase in cell number. With regard to the general problem of shoot histogenesis, our data for the rosette plants and those for Xanthium and Chrysanthemum showing extensive cell division far below the apical meristem, are in full agreement with the studies by Bindloss (1942) with tomato, and support her conclusion that “. . . it is no longer possible to think that the chief center of cell division is in a relatively short zone 60 to 100 microns from the stem tip . . . and that cell division activity in the promeristem is not solely responsible for stem length.” On the contrary, the mitotic activity in the subapical regions is undoubtedly responsible for the major part of the cells found in the stem.     

PHOTOCONTROL OF THE ORIENTATION OF CELL DIVISION IN ADIANTUM. I. EFFECTS OF THE DARK AND RED PERIODS IN THE APICAL CELL OF GAMETOPHYTES

When protonemata of Adiantum capillus-veneris L. which had been grown filamentously under continuous red light were transferred to continuous white light, the apical cell divided transversely twice, but the 3rd division was longitudinal. An intervening period of darkness lasting from 0 to 90 hr either between the 1st and the 2nd cell division or between the 2nd and the 3rd one did not affect the number of protonemata in which the 3rd cell division was longitudinal. The insertion of red light instead of darkness greatly decreased the percentage of 1st longitudinal divisions occurring at the 3rd division, and increased the number of transverse divisions. Fifty percent reduction of induction of 1st longitudinal division was caused by ca. 50 hr exposure to red light between 1st and 2nd division and by ca. 20 hr between 2nd and 3rd division, and total loss was induced by an exposure of ca. 100 hr or longer to red light in the former and by ca. 40 hr longer in the latter. Thus, by using an appropriate intervening dark period or exposure to red light, the orientation and timing of cell division could be controlled in apical cell of the fern protonemata.     

ANALYSIS OF THE GROWTH KINETICS OF A HUMAN LYMPHOMA CELL LINE

The growth kinetics of an established human lymphoma cell line were analyzed by a variety of techniques utilizing various cell inocula (5 x 10⁴ - 5 x 10⁵ cells) dispensed into 60 mm diameter dishes. Techniques included pulse-labeled mitosis (PLM), continuous labeling with ³H-TdR, time-lapse photography (TLP), cell counts by electronic particle counter, and DNA histography obtained by pulse cytophotometry (PCP). There were no significant differences among values determined for any kinetic parameters as a function of cell concentration. the average doubling time of exponentially growing cells, regardless of cell inoculum, was 44.1 hr. the generation time determined by PLM was 31.1 hr with a SD of 4.7 hr. Transit times for each stage were: T_G1= 10.6 hr, T_s= 9.9 hr, T_G2= 9.9 hr, and T_m= 0.7 hr. Repeated experiments using continuous labeling with ³H-TdR demonstrated a T_G2 of 6.3 hr. the longer value determined by PLM is possibly due to the technical manipulations of this procedure which may delay pulse-labeled cells from resuming cell cycle transit. Hence, values for cell cycle stages were recalculated to give T_G1= 14.1 hr, T_s= 9.9 hr, T_G2 = 6.3 hr, and T_m= 0.7 hr. These results were used to compute the size of each cell cycle stage compartment pool and corresponded very closely to values defined directly by PCP. TLP analysis considered only cells that produced colonies of at least thirty-two cells. Generation times ranged from 8 to 89 hr and showed a positive skewness. the average value measured for 330 divisions was 34.5 hr with a SD of 13.2 hr. Thus, the variance predicted by curve fitting of the PLM data did not correlate with that defined by time-lapse photography nor did it encompass the range in generation times observed directly by TLP. There was a positive correlation between sister-sister cell generation times (+0.66) but no relation was noted for mother-daughter values.     

APPLICATION OF THE 22.5 MEV DEUTERON MICROBEAM TO THE STUDY OF MORPHOGENETIC PROBLEMS WITHIN THE SHOOT APEX OF OSMUNDA CLAYTONIANA

Excised shoot apices of Osmunda claytoniana were grown under controlled sterile conditions. Histological examination of the normal shoot apex shows that it is comprised of: (1) a promeristem, which possesses 1 or more apical initiating cells at its center; (2) a prestelar tissue consisting of an incipient vascular tissue which flanks the pith-mother-cell zone; the pith-mother-cell zone gives rise to the pith rib meristem and subsequently to the fundamental parenchyma of the pith; (3) the fundamental parenchyma of the cortex and the fundamental parenchyma of the dermal system both arising from flank cells of the promeristem. Apical initial cells of meristems irradiated with a 127,000 rad acute exposure of a deuteron beam having a diameter of 25μ, histologically examined at 7-day intervals for a 12-week period, as early as 3 weeks’ postirradiation, showed the apical initiating cell(s) together with certain of the cells of the pith-mother-cell zone to be destroyed. A wound response develops peripherally to the destroyed initials. In addition, an isolated, organized growth center is observed to develop from normal promeristem cells. Incipient vascular tissue and a new pith-mother-cell zone are also observed to develop in association with the new center of growth. Implications of the role of the interrelationships between apical initiating cell(s) and other cells of the meristem and the role they may play in maintenance of meristematic integrity within the shoot meristem are discussed.     

QUANTITATIVE STUDIES OF THE VEGETATIVE SHOOT APEX OF EQUISETUM SCIRPOIDES

Equisetum scirpoides Michx., propagated from a single clone, was grown in a controlled growth chamber at 24 ± 1 C under a photoperiod of 16 hr light/8 hr darkness. The apical cell of aerial vegetative shoots gives rise to derivatives (merophytes) in a helical sequence. Each newly formed merophyte divides anticlinally to form two superposed cells that are parallel to a lateral face of the apical cell. Radial longitudinal divisions then take place in the two superposed cells. Shoot tips were fixed every 2 hr for 24 hr to determine the mitotic index of the apical cell, six subjacent cells, and the remaining cells above the level of leaf initiation. Average mitotic indices for the 24-hr period were 3.9%, 3.9%, and 7.0%, respectively. The results indicate that the apical cell is quite active mitotically; there was no clear evidence of endopolyploidy in cells of the shoot apex, young leaves or in the developing cortex, based upon cytophotometric measurements of DNA content.