The Effect of Growth Hormones on Cell Division and Expansion in Liquid Suspension Cultures of Acer pseudoplatanus

The effects on cell division and cell size of indole-3-aceticacid (IAA), gibberellic acid (GA), and kinetin were studiedin liquid suspension cultures of cambial cells derived fromAcer pseudoplatanus. It was shown that all three hormones promotecell division and that the effects of both GA and kinetin areadditive to those of IAA, but the effects of GA and kinetintogether are not additive. Treatment with IAA resulted in anincrease of mean cell size (indicating that cell expansion ispromoted), but after GA or kinetin treatment the mean cell sizewas smaller, indicating that little cell expansion had takenplace after each division. The results are discussed in relationto previous work on the effects of hormones in the intact cambiumand to current theories on the interactions of growth hormones.     

Regulation of Brassica rapa chloroplast proliferation in vivo and in cultured leaf disks

Summary. To understand the regulatory mechanisms of chloroplast proliferation, chloroplast replication was studied in cultured leaf disks cut from plants of 25 species. In leaf disks from Brassica rapa var. perviridis, the number of chloroplasts per cell increased remarkably in culture. We examined chloroplast replication in this plant in vivo and in culture media with and without benzyladenine, a cytokinin. In whole plants, leaf cells undergo two phases from leaf emergence to full expansion: an early proliferative stage, in which mitosis occurs, and a differentiational stage after mitosis has diminished. During the proliferative stage, chloroplast replication keeps pace with cell division. In the differentiational phase, cell division ceases but chloroplast replication continues for two or three more cycles, with the number of chloroplasts per cell reaching about 60. In the leaf disks, the number of chloroplasts per cell increased from about 18 to 300 without benzyladenine, and to over 600 with benzyladenine, indicating that this cytokinin enhances chloroplast replication in cultured tissue. We also studied changes in ploidy and cell volume between in vivo cells and cells grown in culture with and without benzyladenine. Ploidy and cell volume increased in a manner very similar to that of the number of chloroplasts, suggesting a relationship between these phenomena.Correspondence and reprints: Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113-0033, Japan.     

Stunted plant 1 mediates effects of cytokinin, but not of auxin, on cell division and expansion in the root of Arabidopsis

Plants control organ growth rate by adjusting the rate and duration of cell division and expansion. Surprisingly, there have been few studies where both parameters have been measured in the same material, and thus we have little understanding of how division and expansion are regulated interdependently. We have investigated this regulation in the root meristem of the stunted plant 1 (stp1) mutation of Arabidopsis, the roots of which elongate more slowly than those of the wild type and fail to accelerate. We used a kinematic method to quantify the spatial distribution of the rate and extent of cell division and expansion, and we compared stp1 with wild type and with wild type treated with exogenous cytokinin (1 microM zeatin) or auxin (30 nM 2,4-dichlorophenoxyacetic acid). All treatments reduced average cell division rates, which reduced cell production by the meristem. Auxin lowered root elongation by narrowing the elongation zone and reducing the time spent by a cell in this zone, but did not decrease maximal strain rate. In addition, auxin increased the length of the meristem. In contrast, cytokinin reduced root elongation by lowering maximal strain rate, but did not change the time spent by a cell within the elongation zone; also, cytokinin blocked the increase in length and cell number of the meristem and elongation zone. The cytokinin-treated wild type phenocopied stp1 in nearly every detail, supporting the hypothesis that cytokinin affects root growth via STP1. The opposite effects of auxin and cytokinin suggest that the balance of these hormones may control the size of the meristem.     

The Effect of Nutritional Factors and Certain Growth Substances on the Growth of Disks Cut from Young Leaves of Phaseolus

The roles of some chemical factors influencing leaf expansion were investigated using disks cut from the primary leaves of young plants of Phaseolus grown in subdued light. Mineral nutrients, cobalt, sucrose, GA and IAA or NAA at suitable concentrations all caused increases in fresh and dry weights of such disks. When all these substances were applied together the increases in diameter and in fresh and dry weight and cell number were very large and comparable with the rates found in intact leaf tissue. The response of disks to sucrose was found to be light dependent, and a number of other significant interactions were noted. Disks cut from older leaves, in which cell division had ceased, did not show large increases in fresh weight in response to treatment with sucrose, and in this such disks differ from those cut from leaves in which cell divisions are continuing. The possible significance of this is discussed and the roles of light and the other chemical factors investigated are assessed in terms of influence on cell division and expansion in disk tissue.     

Benzyladenine-Enhanced Growth of Attached Young Bean Leaves. Studies with Inhibitors of Nucleic Acid and Protein Synthesis

Benzyladenine (BA) stimulated division but not expansion ofmesophyll cells and repressed chlorophyll accumulation in attachedyoung bean leaves. Even in the presence of fluorodeoxyuridine(FUdR) or mitomycin C which causes complete suppression of BA-inducedincrease in DNA content, BA increased RNA and protein contentsand fresh weight, but decreased chlorophyll accumulation. Moreover,BA n the presence of FUdR induced marked cell expansion. Inthe presence of a-amanitin (AM), BA did not produce any changein DNA content, fresh weight or cell size. All of the BA effectswere observed even in the presence of fluorouracil (FU) plusthymidine (TdR). AM and cycloheximide added 0–12 h effectively inhibitedBA-stimulated cell division but showed no effect if added at18 h. FU plus TdR added 0–18 h had almostno effect onthe cell number at 24 h. These results indicate that BA stimulates the mRNA synthesisnecessary for induction of cell division, and that the synthesisof cytoplasmic rRNA is not always necessary for BA-stimulatedcell division, and moreover, that BA stimulates expansion growthof cells in which DNA synthesis is suppressed. (Received August 16, 1982; Accepted March 31, 1983)     

Unequal cell division, growth regulation and colony size of mammalian cells: a mathematical model and analysis of experimental data.

This work describes mathematically the dynamics of expansion of cell populations from the initial division of single cells to colonies of several hundred cells. This stage of population growth is strongly influenced by stochastic (random) elements including, among others, cell death and quiescence. This results in a wide distribution of colony sizes. Experimental observations of the NIH3T3 cell line as well as for the NIH3T3 cell line transformed with the ras oncogene were obtained for this study. They include the number of cells in 4-day-old colonies initiated from single cells and measurements of sizes of sister cells after division, recorded in the 4-day-old colonies. The sister cell sizes were recorded in a way which enabled investigation of their interdependence. We developed a mathematical model which includes cell growth and unequal cell division, with three possible outcomes of each cell division: continued cell growth and division, quiescence, and cell death. The model is successful in reproducing experimental observations. It provides good fits to colony size distributions for both NIH3T3 mouse fibroblast cells and the same cells transformed with the rasEJ human cancer gene. The difference in colony size distributions could be fitted by assuming similar cell lifetimes (12-13 hr) and similar probabilities of cell death (q = 0.15), but using different probabilities of quiescence, r = 0 for the ras oncogene transformed cells and r = 0.1 for the non-transformed cells. The model also reproduces the evolution of distributions of sizes of cells in colonies, from a single founder cell of any specified size to the stable limit distribution after eight to ten cell divisions. Application of the model explains in what way both random events and deterministic control mechanisms strongly influence cell proliferation at early stages in the expansion of colonies.     

Expression of genomic AtCYCD2;1 in Arabidopsis induces cell division at smaller cell sizes: implications for the control of plant growth

The Arabidopsis (Arabidopsis thaliana) CYCD2;1 gene introduced in genomic form increased cell formation in the Arabidopsis root apex and leaf, while generating full-length mRNA, raised CDK/CYCLIN enzyme activity, reduced G1-phase duration, and reduced size of cells at S phase and division. Other cell cycle genes, CDKA;1, CYCLIN B;1, and the cDNA form of CYCD2;1 that produced an aberrantly spliced mRNA, produced smaller or zero increases in CDK/CYCLIN activity and did not increase the number of cells formed. Plants with a homozygous single insert of genomic CYCD2;1 grew with normal morphology and without accelerated growth of root or shoot, not providing evidence that cell formation or CYCLIN D2 controls growth of postembryonic vegetative tissues. At the root apex, cells progressed normally from meristem to elongation, but their smaller size enclosed less growth and a 40% reduction in final size of epidermal and cortical cells was seen. Smaller elongated cell size inhibited endoreduplication, indicating a cell size requirement. Leaf cells were also smaller and more numerous during proliferation and epidermal pavement and palisade cells attained 59% and 69% of controls, whereas laminas reached normal size. Autonomous control of expansion was therefore not evident in abundant cell types that formed tissues of root or leaf. Cell size was reduced by a greater number formed in a tissue prior to cell and tissue expansion. Initiation and termination of expansion did not correlate with cell dimension or number and may be determined by tissue-wide signals acting across cellular boundaries.     

Division of cell nuclei, mitochondria, plastids, and microbodies mediated by mitotic spindle poles in the primitive red alga Cyanidioschyzon merolae

To understand the cell cycle, we must understand not only mitotic division but also organelle division cycles. Plant and animal cells contain many organelles which divide randomly; therefore, it has been difficult to elucidate these organelle division cycles. We used the primitive red alga Cyanidioschyzon merolae, as it contains a single mitochondrion and plastid per cell, and organelle division can be highly synchronized by a light/dark cycle. We demonstrated that mitochondria and plastids multiplied by independent division cycles (organelle G1, S, G2 and M phases) and organelle division occurred before cell–nuclear division. Additionally, organelle division was found to be dependent on microtubules as well as cell–nuclear division. We have observed five stages of microtubule dynamics: (1) the microtubule disappears during the G1 phase; (2) α-tubulin is dispersed within the cytoplasm without forming microtubules during the S phase; (3) α-tubulin is assembled into spindle poles during the G2 phase; (4) polar microtubules are organized along the mitochondrion during prophase; and (5) mitotic spindles in cell nuclei are organized during the M phase. Microfluorometry demonstrated that the intensity peak of localization of α-tubulin changed in the order to spindle poles, mitochondria, spindle poles, and central spindle area, but total fluorescent intensity did not change remarkably throughout mitotic phases suggesting that division and separation of the cell nucleus and mitochondrion is mediated by spindle pole bodies. Inhibition of microtubule organization induced cell–nuclear division, mitochondria separation, and division of a single membrane-bound microbody, suggesting that similar to cell–nuclear division, mitochondrion separation and microbody division are dependent on microtubules.     

Response of cassava leaf area expansion to water deficit: cell proliferation, cell expansion and delayed development

BACKGROUND AND AIMS: Cassava (Manihot esculenta) is an important food crop in the tropics that has a high growth rate in optimal conditions, but also performs well in drought-prone climates. The objectives of this work were to determine the effects of water deficit and rewatering on the rate of expansion of leaves at different developmental stages and to evaluate the extent to which decreases in cell proliferation, expansion, and delay in development are responsible for reduced growth. METHODS: Glasshouse-grown cassava plants were subjected to 8 d of water deficit followed by rewatering. Leaves at 15 developmental stages from nearly full size to meristematic were sampled, and epidermal cell size and number were measured on leaves at four developmental stages. KEY RESULTS: Leaf expansion and development were nearly halted during stress but resumed vigorously after rewatering. In advanced-stage leaves (Group 1) in which development was solely by cell expansion, expansion resumed after rewatering, but not sufficiently for cell size to equal that of controls at maturity. In Group 2 (cell proliferation), relative expansion rate and cell proliferation were delayed until rewatering, but then recovered partially, so that loss of leaf area was due to decreased cell numbers per leaf. In Group 3 (early meristematic development) final leaf area was not affected by stress, but development was delayed by 4-6 d. On a plant basis, the proportion of loss of leaf area over 26 d attributed to leaves at each developmental stage was 29, 50 and 21 % in Group 1, 2 and 3, respectively. CONCLUSIONS: Although cell growth processes were sensitive to mild water deficit, they recovered to a large extent, and much of the reduction in leaf area was caused by developmental delay and a reduction in cell division in the youngest, meristematic leaves.     

Patterns of cell division and expansion in developing petals of Petunia hybrida

The definition of the patterns of cell division and expansion in plant development is of fundamental importance in understanding the mechanics of morphogenesis. By studying cell division and expansion patterns, we have assembled a developmental map of Petunia hybrida petals. Cycling cells were labelled with in situ markers of the cell cycle, whereas cell expansion was followed by assessing cell size in representative regions of developing petals. The outlined cell division and expansion patterns were related to organ asymmetry. Initially, cell divisions are uniformly distributed throughout the petal and decline gradually, starting from the basal part, to form a striking gradient of acropetal polarity. Cell areas, in contrast, increased first in the basal portion and then gradually towards the petal tip. This growth strategy highlighted a cell size control model based on cell-cycle departure time. The dorso-ventral asymmetry can be explained in terms of differential regulation of cell expansion. Cells of the abaxial epidermis enlarged earlier to a higher final extent than those of the adaxial epidermis. Epidermal appendage differentiation contributed to the remaining asymmetry. On the whole our study provides a sound basis for mutant analyses and to investigate the impact of specific (environmental) factors on petal growth.     

Gibberellin indirectly promotes chloroplast biogenesis as a means to maintain the chloroplast population of expanded cells

Chloroplast biogenesis needs to be well coordinated with cell division and cell expansion during plant growth and development to achieve optimal photosynthesis rates. Previous studies showed that gibberellins (GAs) regulate many important plant developmental processes, including cell division and cell expansion. However, the relationship between chloroplast biogenesis with cell division and cell expansion, and how GA coordinately regulates these processes, remains poorly understood. In this study, we showed that chloroplast division was significantly reduced in the GA‐deficient mutants of Arabidopsis (ga1‐3) and Oryza sativa (d18‐AD), accompanied by the reduced expression of several chloroplast division‐related genes. However, the chloroplasts of both mutants exhibited increased grana stacking compared with their respective wild‐type plants, suggesting that there might be a compensation mechanism linking chloroplast division and grana stacking. A time‐course analysis showed that cell expansion‐related genes tended to be upregulated earlier and more significantly than the genes related to chloroplast division and cell division in GA‐treated ga1‐3 leaves, suggesting the possibility that GA may promote chloroplast division indirectly through impacting leaf mesophyll cell expansion. Furthermore, our cellular and molecular analysis of the GA‐response signaling mutants suggest that RGA and GAI are the major repressors regulating GA‐induced chloroplast division, but other DELLA proteins (RGL1, RGL2 and RGL3) also play a role in repressing chloroplast division in Arabidopsis. Taken together, our data show that GA plays a critical role in controlling and coordinating cell division, cell expansion and chloroplast biogenesis through influencing the DELLA protein family in both dicot and monocot plant species.     

Mechanism for increased leaf growth in elevated CO2

The effect of exposure to elevated CO₂ on the processes of leafcell production and leaf cell expansion was studied using primaryleaves of Phaseolus vulgaris L. Cell division and expansionwere separated temporally by exposing seedlings to dim red lightfor 10 d (when leaf cell division was completed) followed byexposure to bright white light for 14 d (when leaf growth wasentirely dependent on cell expansion). When plants were exposedto elevated CO₂ during the phase of cell expansion, epidermalcell size and leaf area development were stimulated. Three piecesof evidence suggest that this occurred as a result of increasedcell wall loosening and extensibility, (i) cell wall extensibility(WEx, measured as tensiometric extension using an Instron) wassignificantly increased, (ii) cell wall yield turgor (V, MPa)was reduced and (iii) xyloglucan endotransglycosylase (XET)enzyme activity was significantly increased. When plants wereexposed to elevated CO₂ during the phase of cell division, thenumber of epidermal cells was increased whilst final cell sizewas significantly reduced and this was associated with reducedfinal leaf area, WEx and XET activity. When plants were exposedto elevated CO₂ during both phases of cell division and expansion,leaf area development was not affected. For this treatment,however, the number of epidermal cells was increased, but cellexpansion was inhibited, despite exposure to elevated CO₂ duringthe expansion phase. Assessments were also made of the spatialpatterns of WEx across the expanding leaf lamina and the datasuggest that exposure to elevated CO₂ during the phase of leafexpansion may lead to enhanced extensibility particularly atbasal leaf margins which may result in altered leaf shape. The data show that both cell production and expansion were stimulatedby elevated CO₂, but that leaf growth was only enhanced by exposureto elevated CO₂ in the cell expansion phase of leaf development.Increased leaf cell expansion is, therefore, an important mechanismfor enhanced leaf growth in elevated CO₂, whilst the importanceof increased leaf cell production in elevated CO₂ remains tobe elucidated. Key words: Phaseolus vulgaris L., dwarf beans, elevated CO², biophysics of cell expansion, xyloglucan endotransglycosylase, XET, water relations     

Polyploidy and cellular mechanisms changing leaf size: comparison of diploid and autotetraploid populations in two species of Lolium

BACKGROUND AND AIMS: Growth and development of plant organs, including leaves, depend on cell division and expansion. Leaf size is increased by greater cell ploidy, but the mechanism of this effect is poorly understood. Therefore, in this study, the role of cell division and expansion in the increase of leaf size caused by polyploidy was examined by comparing various cell parameters of the mesophyll layer of developing leaves of diploid and autotetraploid cultivars of two grass species, Lolium perenne and L. multiflorum. METHODS: Three cultivars of each ploidy level of both species were grown under pot conditions in a controlled growth chamber, and leaf elongation rate and the cell length profile at the leaf base were measured on six plants in each cultivar. Cell parameters related to division and elongation activities were calculated by a kinematic method. KEY RESULTS: Tetraploid cultivars had faster leaf elongation rates than did diploid cultivars in both species, resulting in longer leaves, mainly due to their longer mature cells. Epidermal and mesophyll cells differed 20-fold in length, but were both greater in the tetraploid cultivars of both species. The increase in cell length of the tetraploid cultivars was caused by a faster cell elongation rate, not by a longer period of cell elongation. There were no significant differences between cell division parameters, such as cell production rate and cell cycle time, in the diploid and tetraploid cultivars. CONCLUSION: The results demonstrated clearly that polyploidy increases leaf size mainly by increasing the cell elongation rate, but not the duration of the period of elongation, and thus increases final cell size.     

Cell size, cell cycle and transition probability in mouse fibroblasts

This paper describes the relationship between cell size and cell division in two situations. In the first, quiescent cells were sorted on the basis of cell size using a fluorescence-activated cell sorter and returned to culture. The results of this type of experiment are compatible with the idea that once cells have completed a size-dependent lag, the rate of entry of cells into S phase is controlled by a rate-limiting random event (or transition).The second kind of experiment follows the kinetics of complete cell cycles in rapidly proliferating cells whose mothers had been sorted on the basis of cell size. The cells born of small mother cells have longer cycle times than cells derived from large mothers. The difference in the cycle time of these two classes was due to differences in the B phase of the cell cycle [containing S, G2, M and part of G1 (G1B)], transition probability being the same in both size classes. Our results show that S, G2 and M are unaffected by size, thus confining the effect of size to G1B. It seems probable that the variability of B phase in cloned cell populations is partly due to variations of cell size at division, and correlations between the cycle times of sister cells result because sibling cells are more similar in size than unrelated cells. The major factor controlling cell division in mouse fibroblasts is shown, however, to be the transition probability; size has a more minor role.     

Morphogenetic Effects on Vegetative Plants of Poa pratensis L. of 6-Azauracil, (2-Chloroethyl)- phosphonic acid, and (2-Chloroethyl) trimethyl-ammonium chloride and their Interaction with Gibberellic Acid

Fresh and dry weights and leaf size of Poa pratensis were reducedwhen treated with 6-azauracil (AzU), (2-chloroethyl)phosphonicacid (CEPA), or (2-chloroethyl)trimethylammonium chloride (CCC).AzU and CEPA inhibited epidermal cell division without inhibitingcell elongation, while CCC inhibited mainly cell elongationand cell division to a small extent. The ratio of blade lengthto sheath length and the blade length/width ratio were reduced,but leaf emergence and tillering were increased by AzU and CEPA.CCC affected only the latter three features. Like GA₃, CEPAinduced stem formation, but internodes were shorter. GA₃ was ineffective in preventing leaf-growth inhibition byAzU, which inhibited Ga₃-induced cell elongation. The inhibitoryeffect of CEPA on leaf growth was apparently reversed by GA₃,but this was due solely to increased cell elongation, the reductionin cell number being unaffected. Ga₃ reversed the effect ofCCC on leaf length, as well as on cell size and number. Simultaneousapplication of the inhibitors produced a complex interactionin reducing leaf length and number and size of epidermis cells.It is postulated that AzU, CEPA, and CCC have different modesof action because they have specific effects on plant growthand different effects on GA₃-induced cell elongation.     

Spatial and temporal effects of free-air CO2 enrichment (POPFACE) on leaf growth,cell expansion,and cell production in a closed canopy of poplar

Leaf expansion in the fast-growing tree, Populus x euramericana was stimulated by elevated [CO(2)] in a closed-canopy forest plantation, exposed using a free air CO(2) enrichment technique enabling long-term experimentation in field conditions. The effects of elevated [CO(2)] over time were characterized and related to the leaf plastochron index (LPI), and showed that leaf expansion was stimulated at very early (LPI, 0-3) and late (LPI, 6-8) stages in development. Early and late effects of elevated [CO(2)] were largely the result of increased cell expansion and increased cell production, respectively. Spatial effects of elevated [CO(2)] were also marked and increased final leaf size resulted from an effect on leaf area, but not leaf length, demonstrating changed leaf shape in response to [CO(2)]. Leaves exhibited a basipetal gradient of leaf development, investigated by defining seven interveinal areas, with growth ceasing first at the leaf tip. Interestingly, and in contrast to other reports, no spatial differences in epidermal cell size were apparent across the lamina, whereas a clear basipetal gradient in cell production rate was found. These data suggest that the rate and timing of cell production was more important in determining leaf shape, given the constant cell size across the leaf lamina. The effect of elevated [CO(2)] imposed on this developmental gradient suggested that leaf cell production continued longer in elevated [CO(2)] and that basal increases in cell production rate were also more important than altered cell expansion for increased final leaf size and altered leaf shape in elevated [CO(2)].     

Effect of indol-3-ylbutyric acid,kinetin and gibberellic acid on alkaloid content inNicotiana rustica stem cuttings

The effect of the growth substances indol-3-ylbutyric acid (IBA), kinetin (Kn) and gibberellic acid (GA3) on the alkaloid content and recovery from the stress induced by removal of roots inNicotiana rustica L. plants have been studied. All treatments, mainly GA₃, overcome the stress more effectively than the controls. The alkaloid content was increased 2.5fold with GA₃. Treatment with Kn also increased the alkaloid content, but only in the absence of roots. In contrast, treatment with IBA slightly reduced the alkaloid content. The results of this investigation indicated that a degradation of stored alkaloids did not occur in spite of the stress.