Restoration of cell growth and proliferation in the wheat roots after their inhibition by nickel sulfate

The dynamics of cell growth and proliferation restoration in different tissues and quiescent center (QC) in the wheat (Triticum aestivum L.) seedling roots and also the differentiation of rhizodermal cells and lateral root initiation after 48-h treatment with 100 μM NiSO₄ were studied. Within 24 h after nickel removal from medium, root growth was resumed due to the increase in the rate of cell growth in the meristem and the region where cell elongation started in control roots. Stimulation of cell proliferation was restored in the main part of the meristem and later in the initial cells of the files and QC. Cell proliferation was not observed in the QC. The time of cell proliferation resumption in the roots and in tested tissues depended on the degree of their injury by nickel treatment. In most tested roots, DNA synthesis and cell division were restored in 32 h. In the cells leaving the meristem due to the resumption of their growth and proliferation, growth of root hairs started. In 48 h, the number of roots with perished cells in the rhizodermis in the meristem was sharply increased and the regeneration of the damaged region by the cells of outer cortex was observed. Only after the appearance of root hairs, the cells coming from the meristem started to elongate. In most roots, the formation of the new elongation zone occurred in 56 h. During its formation, the initiation of lateral root primordia was shifted in the basipetal direction. It was concluded that the cessation of cell growth and proliferation under the influence of high concentration of heavy metal (HM) ions is not lethal for the root. At the action of toxic HM concentrations, the plant strategy is the maintenance of meristematic cell capacity for cell growth and proliferation resumption. The cellular mechanism of this capacity maintenance is the transition of meristematic cells from G₁ phase to dormancy due to growth inhibition and the inhibition of the transition to DNA synthesis.     

Meristem as a Self-Renewing System: Maintenance and Cessation of Cell Proliferation (A Review)

The mechanisms of the maintenance of long-term cell proliferation and its cessation in the meristem of the growing root were analyzed. Quiescent center (QC) remains in the meristem for a long time, whereas all other cells leave the meristem after several mitotic cycles. The question arises as to what extent such organization of proliferation corresponds to the concept of stem cells elaborated for animals. The definition of animal stem cells is met by the QC cells rather than by actively dividing cells that boundary it. However, QC is not a self-maintaining population of cells originated during early stages of embryogenesis; it is formed from dividing cells in the main or lateral root. After root decapitation, the QC can arise from the cells that normally would leave the meristem before long. There is a zone of the meristem whose cells are capable of remaining and forming QC after the removal of the apical part of the root. Maintenance of the size of the meristem depends on the interaction between QC, initial cells located at its surface, and the actively dividing cells. Apparently, the life span of cells in the meristem determines the time when the meristematic cell will begin the elongation. The number of cells starting the elongation depends on proliferation rate and on the changes in life span of meristematic cells which determine their initial number. The life span of the cells in the meristem for most actively dividing cells does not depend on the cell divisions, and remains unchanged in the presence of various inhibitors. As a result of inhibited proliferation in the main part of the meristem, cell divisions in the QC are activated and newly formed cells may proceed to rapid divisions. Thus, the size of the meristem is maintained by the operation of several mechanisms, and individual processes may be, on the one hand, relatively independent and, on the other hand, regulated either by feedback or directly. As a result, the root growth becomes resistant to various external events.     

Using the roots as test objects for the assessment of biological action of chemical substances

A sound approach to the root usage as model objects for the assessment of biological activity of chemical substances and environmental stressors is suggested on the basis of the analysis of various inhibitor and radiation action on the root. It is analyzed on the cellular level, how steady growth is maintained under various stress action. Special attention is paid to the meristematic cell transition to elongation, which is controlled by the two groups of processes: the first ones determine the rate of cell proliferation and the second ones determine the cell life span in the meristem. The rate of cell proliferation is rather sensitive to various treatments; in contrast, the processes controlling the cell life span in the meristem are rather stable. It is shown that studying the kinetics of the root growth rate gives much more information than a single measurement of root length increment. A possibility of root usage for the search of efficient cytostatics is exemplified. The role of the quiescent center in growth resumption after various stressful treatments is considered.     

The influence of 2,4-dichlorophenoxyacetic acid on cell cycle Kinetics and Sister-Chromatid Exchange Frequency in Garlic (Allium sativum L.) Meristem Cells

The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) at low concentrations on cell cycle duration and sister-chromatid exchange (SCE) frequency was studied using meristem root-tip cells ofAllium sativum L. 2,4-D induced a marked prolongation of the cell cycle. At the same time, small but statistically significant increases in SCE frequencies were observed at 5 μM and 15 μM 2,4-D concentrations. The significance of these findings in the evaluation of mutagenic activity of 2,4-D is discussed.     

生长素类化合物及6－苯甲基腺嘌呤对拟南芥主根生长的抑制效应比较

为更好的研究生长素类化合物及6-苯甲基腺嘌呤（6－BA）对细胞分裂和细胞伸长的影响,以拟南芥主根为材料,从组织学水平比较了IAA、NAA、2,4－D和6－BA对拟南芥主根分生区和伸长区的抑制效应,发现IAA和NAA效果是相似的,可以通过促进细胞分裂显著增加根分生区长度,但也显著缩短主根仲长区长度,而2,4－D和6－BA则通过抑制细胞分裂来显著缩短根分生区长度,同时也显著缩短根伸长区的长度。     

The Root Apex of Arabidopsis thaliana Consists of Four Distinct Zones of Growth Activities: Meristematic Zone,Transition Zone,Fast Elongation Zone and Growth Terminating Zone

In the growing apex of Arabidopsis thaliana primary roots, cells proceed through four distinct phases of cellular activities. These zones and their boundaries can be well defined based on their characteristic cellular activities. The meristematic zone comprises, and is limited to, all cells that undergo mitotic divisions. Detailed in vivo analysis of transgenic lines reveals that, in the Columbia-0 ecotype, the meristem stretches up to 200 µm away from the junction between root and root cap (RCJ). In the transition zone, 200 to about 520 µm away from the RCJ, cells undergo physiological changes as they prepare for their fast elongation. Upon entering the transition zone, they progressively develop a central vacuole, polarize the cytoskeleton and remodel their cell walls. Cells grow slowly during this transition: it takes ten hours to triplicate cell length from 8.5 to about 35 µm in the trichoblast cell files. In the fast elongation zone, which covers the zone from 520 to about 850 µm from the RCJ, cell length quadruplicates to about 140 µm in only two hours. This is accompanied by drastic and specific cell wall alterations. Finally, root hairs fully develop in the growth terminating zone, where root cells undergo a minor elongation to reach their mature lengths.Key words: Arabidopsis, cytoskeleton, development, differentiation zone, elongation zone, growth, growth terminating zone, meristem, root apex, transition zone     

Dynamics of growth,proliferation and differentiation of wheat root cells exposed to a high nickel concentration

The dynamics of root growth, proliferation of initial cells of the root cap, rhizodermis, and central metaxylem, as well as structural changes in the cells induced by a 72-h exposure to a high (0.1 mM) concentration of NiSO₄ were studied in 3-day-old wheat (Triticum aestivum L.) seedlings. In the roots of control plants, we observed a 12-h rhythm of changes in the length of the cells that completed elongating. Upon the treatment with nickel, this effect was negated, and a considerable reduction in the root length increment was observed in 12 h. In 24 h, root growth essentially ceased. Cell elongation was suppressed acropetally, and the cells, whose elongation was over, became shorter. In the meristem and apical part of the elongation zone, slow cell growth continued during the second and even third days. Autoradiography showed that the earliest effect of nickel on the processes of root morphogenesis observed in 6 h was a suppression of cell transition to DNA synthesis. The cells, where DNA synthesis has already started or which were in other stages of the cycle, continued to pass slowly through the cycle and completed it. Sister cells formed as a result of division subsequently left the cycle in the phase G1 and transited to dormancy. It was found that the main mechanism of cell proliferation cessation was the suppression of cell transition to DNA synthesis. In the cells elongating when exposed to nickel, tissue-specific changes in the nucleus structure were observed (chromatolysis in the rhizodermis and cortex, pycnosis in the endodermis, a disturbance of the nucleus structure in the central metaxylem). These disorders were only observed after cessation of elongation. Root incubation in 0.1 mM nickel solution did not affect the onset of cell differentiation in the xylem and metaphloem and shifted its beginning to the root tip. However, in 24 h the initiation and growth of root hairs were suppressed. It was concluded that tissue-specific nickel-induced changes in the nucleus structure in the elongating cells do not cause the cessation of root growth, although point to nickel toxic effect on the cells in the course of elongation.     

Brassinosteroids control meristem size by promoting cell cycle progression in Arabidopsis roots

Brassinosteroids (BRs) play crucial roles in plant growth and development. Previous studies have shown that BRs promote cell elongation in vegetative organs in several plant species, but their contribution to meristem homeostasis remains unexplored. Our analyses report that both loss- and gain-of-function BR-related mutants in Arabidopsis thaliana have reduced meristem size, indicating that balanced BR signalling is needed for the optimal root growth. In the BR-insensitive bri1-116 mutant, the expression pattern of the cell division markers CYCB1;1, ICK2/KRP2 and KNOLLE revealed that a decreased mitotic activity accounts for the reduced meristem size; accordingly, this defect could be overcome by the overexpression of CYCD3;1. The activity of the quiescent centre (QC) was low in the short roots of bri1-116, as reported by cell type-specific markers and differentiation phenotypes of distal stem cells. Conversely, plants treated with the most active BR, brassinolide, or mutants with enhanced BR signalling, such as bes1-D, show a premature cell cycle exit that results in early differentiation of meristematic cells, which also negatively influence meristem size and overall root growth. In the stem cell niche, BRs promote the QC renewal and differentiation of distal stem cells. Together, our results provide evidence that BRs play a regulatory role in the control of cell-cycle progression and differentiation in the Arabidopsis root meristem.     

Further biological characteristics of galactoglucomannan oligosaccharides

The biological activity of cell wall-derived galactoglucomannan oligosaccharides (GGMOs) was dependent on their chemical structure. Galactosyl side chains linked to the glucomanno-core influenced their inhibition of elongation growth of pea (Pisum sativum L. cv. Tyrkys) stem segments induced by 2,4-dichlorophenoxyacetic acid (2,4-D). Reduction of the number of galactosyl side chains in GGMOs caused stimulation of the endogenous growth. Modification on the glucomanno-reducing end did not affect significantly the activity of these oligosaccharides. GGMOs inhibited also the elongation induced by indole-3-acetic acid (IAA) and gibberellic acid (GA₃). In the presence of IAA the elongation growth was inhibited to 20 – 35 % after 24 h of incubation depending on GGMOs concentrations (1 μM, 10 nM, 0.1 nM), similarly as in the presence of 2,4-D, which confirms the hypothesis of GGMOs antiauxin properties. The elongation induced by GA₃ was inhibited to 25 – 60 %, however, the time course of inhibition was different compared with IAA and 2,4-D. The highest inhibition was determined already after 6 h of incubation with a significant decrease after this time. The results indicated a competition between GGMOs and growth regulators.     

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.     

Root growth, developmental changes in the apex, and hydraulic conductivity for Opuntia ficus-indica during drought

Developmental changes in the root apex and accompanying changes in lateral root growth and root hydraulic conductivity were examined for Opuntia ficus-indica (L.) Miller during rapid drying, as occurs for roots near the soil surface, and more gradual drying, as occurs in deeper soil layers. During 7 d of rapid drying (in containers with a 3-cm depth of vermiculite), the rate of root growth decreased sharply and most root apices died; such a determinate pattern of root growth was not due to meristem exhaustion but rather to meristem mortality after 3 d of drying. The length of the meristem, the duration of the cell division cycle, and the length of the elongation zone were unchanged during rapid drying. During 14 d of gradual drying (in containers with a 6-cm depth of vermiculite), root mortality was relatively low; the length of the elongation zone decreased by 70%, the number of meristematic cells decreased 30%, and the duration of the cell cycle increased by 36%. Root hydraulic conductivity ( L _P) decreased to one half during both drying treatments; L _P was restored by 2 d of rewetting owing to the emergence of lateral roots following rapid drying and to renewed apical elongation following gradual drying. Thus, in response to drought, the apical meristems of roots of O. ficus-indica near the surface die, whereas deeper in the substrate cell division and elongation in root apices continue. Water uptake in response to rainfall in the field can be enhanced by lateral root proliferation near the soil surface and additionally by resumption of apical growth for deeper roots.     

A model for dynamics of cell division cycle in onion roots

Summary The study of the cell division cycle by means of caffeine labelling inAllium roots, at 15° C, employing intact root and decapitated roots at several levels (0.5, 1.0, 1.5, 2.0, and 2.5 mm) has shown that the number of cycles developed by the cells is constant at each meristem level. This number and the durations of the cycles are not affected by the decapitation. It is suggested that the cell cycle is controlled in the meristematic cells by an intracellular programme which would be developed throughout the meristem.However, the larger the region decapitated is, the more decreases the growth rate of the roots. The removal of the root cap (about 0.5 mm) did not modify the rate of root growth, although it blocked the geotropic response. The quiescent center is proposed as a source of auxin controlling cell elongation.     

Auxin, actin and growth of the Arabidopsis thaliana primary root

To understand how auxin regulates root growth, we quantified cell division and elemental elongation, and examined actin organization in the primary root of Arabidopsis thaliana. In treatments for 48 h that inhibited root elongation rate by 50%, we find that auxins and auxin-transport inhibitors can be divided into two classes based on their effects on cell division, elongation and actin organization. Indole acetic acid (IAA), 1-naphthalene acetic acid (NAA) and tri-iodobenzoic acid (TIBA) inhibit root growth primarily through reducing the length of the growth zone rather than the maximal rate of elemental elongation and they do not reduce cell production rate. These three compounds have little effect on the extent of filamentous actin, as imaged in living cells or by chemical fixation and immuno-cytochemistry, but tend to increase actin bundling. In contrast, 2,4-dichlorophenoxy-acetic acid (2,4-D) and naphthylphthalamic acid (NPA) inhibit root growth primarily by reducing cell production rate. These compounds remove actin and slow down cytoplasmic streaming, but do not lead to mislocalization of the auxin-efflux proteins, PIN1 or PIN2. The effects of 2,4-D and NPA were mimicked by the actin inhibitor, latrunculin B. The effects of these compounds on actin were also elicited by a 2 h treatment at higher concentration but were not seen in two mutants, eir1-1 and aux1-7, with deficient auxin transport. Our results show that IAA regulates the size of the root elongation zone whereas 2,4-D affects cell production and actin-dependent processes; and, further, that elemental elongation and localization of PINs are appreciably independent of actin.     

Dependence of Root Cell Growth and Division on Root Diameter

Primary roots of 98 species from different families of monocotyledonous and dicotyledonous plants and adventitious roots obtained from bulbs and rhizomes of 24 monocot species were studied. Root growth rate, root diameter, length of the meristem and elongation zones, number of meristematic cells in a file of cortical cells, and length of fully elongated cells were evaluated in each species after the onset of steady growth. The mitotic cycle duration and relative cell elongation rate were calculated. In all species, the meristem length was approximately equal to two root diameters. When comparing different species, the rate of root growth increased with a larger root diameter. This was due to an increase in the number of meristematic cells in a row and, to a lesser degree, to a greater length of fully elongated cells. The duration of the mitotic cycle and the relative cell elongation rate did not correlate with the root diameter. It is suggested that the meristem size depends on the level of nutrient inflow from upper tissues, and is thereby controlled during further growth.     

Regions of differential cell elongation and mitosis, and root meristem morphology in different tissues of geotropically stimulated maize root apices

We examined cell length, mitosis, and root meristem “cuticle” in different tissues of geostimulated, red light-exposed primary roots of corn (Zea Mays, Wisconsin hybrid 64A × 22R). The examination was done at 15-minute intervals for a period of 240 minutes. Differences in cell elongation between the upper and lower sides were most prominent between 1.5 and 2.5 mm from the root meristem; the outer cortex had the greatest elongation growth, and the upper cells showed a significant increase in length compared to the lower. A differential mitosis was also found, with the lower tissue being greater. We infer that the mitotic activity is indicative of cell division, and this division occurs strictly in the first 1.5 mm of the root meristem. The combined effect of differential cell elongation and cell division results in the localization of the geotropic curvature in the 1.5- to 2.5-mm region from the root meristem. Mitosis that occurs primarily in the cortex and stele were asynchronous; the peak of cortical division preceded that of the stele. Both peaks occurred before the peak of geotropism. A densely stained layer separates the cap from the root meristem. This layer is thinner at the apex of the root meristem. The area of the thin region increased with time and peaked at 180 minutes after geostimulation, which was coincidental with the peak of the geotropic response.     

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.     

A morphological and histological comparison of the initiation and development of pecan (Carya illinoinensis) somatic embryogenic cultures induced with naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid

Summary Somatic embryos produced in vitro may exhibit structural abnormalities that affect their subsequent germination and conversion into plants. To assess the influence of auxin type on embryo initiation and development, a morphological and histological comparison was made of pecan (Carya illinoinensis) somatic embryogenic cultures induced on media with naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid (2,4-D), using light and scanning electron microscopy. Both auxins promoted enhanced cell division, particularly in subepidermal cell layers. However, notable differences were observed in mitotic activity, location of embryogenic cell proliferation, epidermal continuity, callus growth, and embryo morphology. Cultures induced on naphthaleneacetic acid had embryogenic regions composed of homogeneous, isodiametric, meristematic cells. Embryos derived from these cultures generally had a normal morphology, were single, and had a discrete apical meristem. In contrast, tissues induced on media with 2,4-D had more intense and heterogeneous regions of cell division. Proliferating cell regions were composed of meristematic cells interspersed with callus and involved more extensive regions of the mesophyll. Marked callus proliferation caused epidermal rupture in some areas. Embryos induced on medium with 2,4-D had a higher incidence of abnormalities that included fasciated, fan-shaped, and tubular embryos. Defined apical meristems were often lacking or partially obliterated due to callus proliferation. The heterogeneous, often intensive proliferation of cells in cultures induced with 2,4-D may interfere with normal patterns of embryo development.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - SEM scanning electron microscopy     

Responses of Mentha suspension-cultured cells to 2,4-dichlorophenoxyacetic acid and accumulation of esterified phenolic acids in their cell walls.

Two distinct types of cell growth of suspension-cultured Mentha were formed when the cells maintained in the medium containing 1000 micrograms l-1 2,4-D were subcultured into different 2,4-D concentrations. Few cell elongation of Mentha (average cell length: 34-40 microns) was observed after division in the medium containing 1-200 micrograms l-1 2,4-D; and significant cell elongation (average cell length: 95-130 microns) was observed after cell division in the medium containing 500-2000 micrograms l-1 2,4-D. A close correlation between culture medium and water content in the cells indicated that 2,4-D promoted cell elongation by water uptake. Amounts of phenolic acid in cell walls were much higher in unelongated cell walls than in elongated ones during the cultivation, and there was a close correlation between the amounts and the level of PAL activity in elongated and unelongated cells. However, there was no significant difference in cell wall components and its neutral sugar composition between elongated and unelongated cells.     

The Effects of H<Superscript>+</Superscript>-ATPase Activator and Inhibitors on Cell Growth in the Maize Root

We studied the effects of H⁺-ATPase activator fusicoccin (FC) and its inhibitors, sodium orthovanadate (Na₃VO₄) and diethylstilbestrol (DES), on the rate of proton secretion by root regions located at various distances from the root tip, the rate of root growth, the length of the fully-elongated root cells, the sizes of growth zones, the relative growth rate of cells along the root length, and the number of fully-elongated cells in the root length increment. FC (10⁻⁶ M) stimulated proton secretion by root segments and enhanced root growth due to the greater length of fully-elongated cells. DES (10⁻⁴ M) suppressed proton secretion and retarded root growth, decreased the length of fully-elongated cells, inhibited cell division, and slowed down cell transition to elongation by prolonging the life-span of cells in the meristem. Na₃VO₄ (10⁻³ and 10⁻⁴ M) exerted similar effects. FC, DES, and orthovanadate did not affect the ratio of the relative rate of cell growth in the elongation zone to that in meristem.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 558–565.Original Russian Text Copyright © 2005 by Mesenko, Ivanov.