Effect of 2,4-D on cell proliferation and elongation in the roots of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

We investigated the effect of 2,4-D (2,4-dichlorophenoxyacetic acid) at concentrations of 1.5, 15, 30, and 60 nM on the growth of the main root of 3–7-d-old plants of Arabidopsis thaliana L. On the basis of measurements of the rate of root growth, lenght of fully elongated cells, and the number of cells in the meristem and elongation zone, we calculated the rates of cell proliferation and their transition to elongation, duration of cell cycle, and life span of cells in the meristem. At a concentration of 1.5 nM, 2,4-D did not affect these characteristics. At concentrations above 1.5 nM, 2,4-D noticeably retarded root growth, which was accounted for by a reduction in the length of cells that completed elongation, deceleration of cell proliferation and their transition to elongation, and prolongation of cell cycle and life span of the cells in the meristem. Thus, auxin decelerated root growth not only as a result of suppression of cell elongation but also at the higher concentrations via retardation of cell divisions in the meristem and their transition to elongation.     

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.     

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.     

Different Degree in Proteasome Malfunction Has Various Effects on Root Growth Possibly through Preventing Cell Division and Promoting Autophagic Vacuolization

The ubiquitin/proteasome pathway plays a vital role in plant development. But the effects of proteasome malfunction on root growth, and the mechanism underlying this involvement remains unclear. In the present study, the effects of proteasome inhibitors on Arabidopsis root growth were studied through the analysis of the root length, and meristem size and cell length in maturation zone using FM4–64, and cell-division potential using GFP fusion cyclin B, and accumulation of ubiquitinated proteins using immunofluorescence labeling, and autophagy activity using LysoTracker and MDC. The results indicated that lower concentration of proteasome inhibitors promoted root growth, whereas higher concentration of inhibitors had the opposite effects. The accumulation of cyclin B was linked to MG132-induced decline in meristem size, indicating that proteasome malfunction prevented cell division. Besides, MG132-induced accumulation of the ubiquitinated proteins was associated with the increasing fluorescence signal of LysoTracker and MDC in the elongation zone, revealing a link between the activation of autophagy and proteasome malfunction. These results suggest that weak proteasome malfunction activates moderate autophagy and promotes cell elongation, which compensates the inhibitor-induced reduction of cell division, resulting in long roots. Whereas strong proteasome malfunction induces severe autophagy and disturbs cell elongation, resulting in short roots.     

Constitutive autophagy in plant root cells

In previous studies, using a membrane-permeable protease inhibitor, E-64d, we showed that autophagy occurs constitutively in the root cells of barley and Arabidopsis. In the present study, a fusion protein composed of the autophagy-related protein AtAtg8 and green fluorescent protein (GFP) was expressed in Arabidopsis to visualize autophagosomes. We first confirmed the presence of autophagosomes with GFP fluorescence in the root cells of seedlings grown on a nutrient-sufficient medium. The number of autophagosomes changed as the root cells grew and differentiated. In cells near the apical meristem, autophagosomes were scarcely found. However, a small but significant number of autophagosomes existed in the elongation zone. More autophagosomes were found in the differentiation zone where cell growth ceases but the cells start to form root hair. In addition, we confirmed that autophagy is activated under starvation conditions in Arabidopsis root cells. When the root tips were cultured in a sucrose-free medium, the number of autophagosomes increased in the elongation and differentiation zones, and a significant number of autophagosomes appeared in cells near the apical meristem. The results suggest that autophagy in plant root cells is involved not only in nutrient recycling under nutrient-limiting conditions but also in cell growth and root hair formation.     

多胺生物合成抑制剂D-精氨酸对拟南芥幼苗根系生长的影晌

为探讨多胺生物合成抑制剂D-精氨酸（D-arginine,D-Arg）对拟南芥根系生长的影响,首先用腐胺（0．1mmol‘L-1）和D—Arg（1．0mmol·L-1）处理种子萌发后生长2d的拟南芥幼苗。腐胺（0．1mmol·L-1）显著促进主根伸长,D-Arg（1．0mmol-L-1）显著抑制主根伸长,并对主根根尖的细胞形态有明显影响。为了进一步了解D—Arg影响拟南芥主根生长的机理,采用浓度梯度D．Arg处理幼苗根系。实验结果表明,随着D-Arg浓度增加（0．2～1．0mmol·L-1）,拟南芥幼苗主根生长受抑制的程度越严重。微分干涉观察主根根尖发现,外源施加D—Arg,引起拟南芥主根根尖分生区的细胞数目减少,使拟南芥幼苗表现出主根的伸长生长变缓。当分生区数目较少时,出现主根几乎不再仲长的现象。由此推测,多胺生物合成抑制剂D-Arg对拟南芥幼苗根生长的抑制作用机制,是D-Arg影响了其根尖分生区的细胞分裂活动,使分生区细胞数目减少,从而引起分生区长度减小,最终导致拟南芥主根仲长生长受到抑制。     

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.     

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.     

Pericycle cell proliferation and lateral root initiation in Arabidopsis

In contrast with other cells generated by the root apical meristem in Arabidopsis, pericycle cells adjacent to the protoxylem poles of the vascular cylinder continue to cycle without interruption during passage through the elongation and differentiation zones. However, only some of the dividing pericycle cells are committed to the asymmetric, formative divisions that give rise to lateral root primordia (LRPs). This was demonstrated by direct observation and mapping of mitotic figures, cell-length measurements, and the histochemical analysis of a cyclin-GUS fusion protein in pericycle cells. The estimated duration of a pericycle cell cycle in the root apical meristem was similar to the interval between cell displacement from the meristem and the initiation of LRP formation. Developmentally controlled LRP initiation occurs early, 3 to 8 mm from the root tip. Thus the first growth control point in lateral root formation is defined by the initiation of primordia in stochastic patterns by cells passing through the elongation and young differentiation zones, up to where lateral roots begin to emerge from the primary root. Therefore, the first growth control point is not restricted to a narrow developmental window. We propose that late LRP initiation is developmentally unrelated to the root apical meristem and is operated by a second growth control point that can be activated by environmental cues. The observation that pericycle cells divide and lateral root primordia form without intervening mitotic quiescence suggests that lateral organ formation in roots and shoots might not be as fundamentally different as previously thought.     

Root Growth Activity of Barban in Relation to Auxin and Other Growth Factors

The effect of barban (4-Cl-2-butynyl-N-3-Cl-phenylcarbamate) on the growth of roots of wheat seedlings has been studied. In concentrations of 10^?7 to 5 · 10^?7M barban causes rapid inhibitions of cytokineses and cell elongation, the effects of which are spontaneously reversible. The reversion of the meristem inhibition is enhanced by thymidylic acid and indole-3-acetic acid (IAA). Initiation of cell elongation is slowed down or ceases during cytostasis; its reversal, on the other hand, is promoted by IAA and kinetin but inhibited by Fe. The final cell elongation attained is strongly reduced by barban and reversed under transient aberrant elongation. This inhibition and the recovery appear both to be additive to cell elongation actions of auxin and antiauxin but reversed by nucleic acid components. The inhibition of elongation is increased by Fe. The following explanation for this phenomenon is suggested: the primary effect of barban is known to be the blocking of metaphases under anaesthesis; this blocking then leads to reduced activation of IAA, kinetin and other metabolites. Auxin is required for cell divisions and initiation of elongation: the apical root growth equals in this respect that of shoot apices and lateral meristems. Initiation of cell elongation is closely dependent upon metabolites produced in dividing meristematic cells, whereas the limitation of cell stretching is independent of the meristem activity. No explanation is offered for the role of Fe.     

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。     

Effects of Surface Area and Flow Rate on Marine Bacterial Growth in Activated Carbon Columns

The colonization of granular activated carbon columns by bacteria can have both beneficial and potentially detrimental consequences. Bacterial growth on the carbon surface can remove adsorbed organics and thus partially regenerate the carbon bed. However, growth can also increase the levels of bacteria in the column effluents, which can adversely affect downstream uses of the treated water. This study of a sand column and several activated carbon columns demonstrated that considerable marine bacterial growth occurred in both sand and carbon columns and that this growth increased the number of bacteria in column effluents. Activated carbon supported approximately 50% more bacteria than did sand. Bacterial growth on activated carbon was reduced by increasing the flow rate through a carbon column and increasing the carbon particle size. Scanning electron micrographs showed that bacteria preferred to attach in the protected crevices on both the sand and carbon surface. The results of this study indicated that the colonization of activated carbon by marine bacteria was enhanced because of carbon's high surface area, its rough surface texture, and its ability to absorb organic materials.     

Temperature‐compensated cell production rate and elongation zone length in the root of Arabidopsis thaliana

To understand how root growth responds to temperature, we used kinematic analysis to quantify division and expansion parameters in the root of Arabidopsis thaliana. Plants were grown at temperatures from 15 to 30 °C, given continuously from germination. Over these temperatures, root length varies more than threefold in the wild type but by only twofold in a double mutant for phytochrome‐interacting factor 4 and 5. For kinematics, the spatial profile of velocity was obtained with new software, Stripflow. We find that 30 °C truncates the elongation zone and curtails cell production, responses that probably reflect the elicitation of a common pathway for handling severe stresses. Curiously, rates of cell division at all temperatures are closely correlated with rates of radial expansion. Between 15 to 25 °C, root growth rate, maximal elemental elongation rate, and final cell length scale positively with temperature whereas the length of the meristem scales negatively. Non‐linear temperature scaling characterizes meristem cell number, time to transit through either meristem or elongation zone, and average cell division rate. Surprisingly, the length of the elongation zone and the total rate of cell production are temperature invariant, constancies that have implications for our understanding of how the underlying cellular processes are integrated.     

Functional identification of AtSKIP as a regulator of the cell cycle signaling pathway in Arabidopsis thaliana

Light is a key environmental cue controlling plant development, which involves meristemic activation by cell proliferation and differentiation. Here, we identify one gene, AtSKIP, associated with cell cycle-regulated root and leaf growth processes in Arabidopsis. The spatial pattern of β-glucuronidase (GUS) activity indicated that AtSKIP is expressed in the leaf primodia, root meristem region and root vascular system, and can be activated by light. Ectopic expression of AtSKIP resulted in enhanced leaf development but suppressed root elongation in Arabidopsis, whereas AtSKIP^DD seedlings displayed retarded leaf growth and normal root growth. Moreover, AtSKIP cells displayed enhanced sensitivity to a cytokinin in a callus induction assay, further demonstrated that AtSKIP expression altered endogenous cell cycle-regulated signaling in plants. Together, these data indicate that AtSKIP participates in cell cycle-mediated growth of leaf and root.     

Analysis of Cell Division and Elongation Underlying the Developmental Acceleration of Root Growth in Arabidopsis thaliana

To investigate the relation between cell division and expansion in the regulation of organ growth rate, we used Arabidopsis thaliana primary roots grown vertically at 20°C with an elongation rate that increased steadily during the first 14 d after germination. We measured spatial profiles of longitudinal velocity and cell length and calculated parameters of cell expansion and division, including rates of local cell production (cells mm⁻¹ h⁻¹) and cell division (cells cell⁻¹ h⁻¹). Data were obtained for the root cortex and also for the two types of epidermal cell, trichoblasts and atrichoblasts. Accelerating root elongation was caused by an increasingly longer growth zone, while maximal strain rates remained unchanged. The enlargement of the growth zone and, hence, the accelerating root elongation rate, were accompanied by a nearly proportionally increased cell production. This increased production was caused by increasingly numerous dividing cells, whereas their rates of division remained approximately constant. Additionally, the spatial profile of cell division rate was essentially constant. The meristem was longer than generally assumed, extending well into the region where cells elongated rapidly. In the two epidermal cell types, meristem length and cell division rate were both very similar to that of cortical cells, and differences in cell length between the two epidermal cell types originated at the apex of the meristem. These results highlight the importance of controlling the number of dividing cells, both to generate tissues with different cell lengths and to regulate the rate of organ enlargement.     

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.     

Cell cycle modulation in the response of the primary root of Arabidopsis to salt stress

Salt stress inhibits plant growth and development. We investigated the importance of cell cycle regulation in mediating the primary root growth response of Arabidopsis to salt stress. When seedlings were transferred to media with increasing concentrations of NaCl, root growth rate was progressively reduced. At day 3 after transfer of seedlings to growth medium containing 0.5% NaCl the primary roots grew at a constant rate well below that prior to the transfer, whereas those transferred to control medium kept accelerating. Kinematic analysis revealed that the growth reduction of the stressed roots was due to a decrease in cell production and a smaller mature cell length. Surprisingly, average cell cycle duration was not affected. Hence, the reduced cell production was due to a smaller number of dividing cells, i.e. a meristem size reduction. To analyze the mechanism of meristem size adaptation prior to day 3, we investigated the short-term cell cycle events following transfer to saline medium. Directly after transfer cyclin-dependent kinase (CDK) activity and CYCB1;2 promoter activity were transiently reduced. Because protein levels of both CDKA;1 and CDKB1;1 were not affected, the temporary inhibition of mitotic activity that allows adaptation to the stress condition is most likely mediated by posttranslational control of CDK activity. Thus, the adaptation to salt stress involves two phases: first, a rapid transient inhibition of the cell cycle that results in fewer cells remaining in the meristem. When the meristem reaches the appropriate size for the given conditions, cell cycle duration returns to its default.     

钙调素参与拟南芥根细胞增殖及幼苗对外源脱落酸的敏感性反应过程

钙调素作为真核细胞的重要信号蛋白,在真核生物正常及逆境条件下的生长发育中发挥着重要作用.研究报道钙调素可促进离体培养的高等动植物细胞的增殖,但有关钙调素蛋白在植物体内的细胞增殖功能尚未见报道.特别是拟南芥基因组中存在7个编码经典钙调素亚型的基因,多数编码基因的功能有待进一步探究.首先借助常用的钙调素拮抗剂W7进行药理学实验,结果表明,野生型拟南芥幼苗根的生长受到了明显的抑制,根尖分生区的面积变小、细胞数目明显减少,根尖分生区中细胞分裂标记基因CYCB1;1的表达受到了明显抑制,这表明在根尖分生区W7可能通过对活性钙调素的抑制作用影响了根尖分生区域的细胞增殖,而根尖分生区正常的细胞增殖需要一定量活性钙调素蛋白的存在.脱落酸(ABA)是植物逆境下的重要激素,在植物种子萌发及幼苗生长发育中发挥着重要作用,W7存在下的拟南芥幼苗对ABA的敏感性下降.借助反向遗传学手段获得了拟南芥中三个编码典型钙调素蛋白基因的三重缺失突变体cam234,蛋白质印迹结果表明三重缺失突变体中钙调素蛋白的含量明显降低.相同培养条件下与野生型相比,三重突变体幼苗根长变短,并且幼苗对ABA敏感性也表现下降趋势,暗示着这三个基因编码的钙调素蛋白可能参与了根分生区域细胞增殖过程及幼苗对脱落酸的敏感性反应,讨论了钙调素的细胞增殖功能及与幼苗对脱落酸的敏感性反应间的关系.     

Localization of organelle DNA synthesis within the root apical meristem of rice

DNA synthesis in cell nuclei and organelles in the root apicalmeristem of rice was analysed by anti-BrdU immunofluorescencemicroscopy to determine whether there is a specific order ofthese events in monocot roots. In the root meristem, organelleDNAs were synthesized in a specific region in the distal partof the root apical meristem, and were not synthesized in theroot meristem‘s proximal region or the elongation zone.In contrast, cell nuclear DNA was synthesized throughout theroot apical meristem, except in the quiescent centre. In theroot cap of rice, DNA synthesis in both cell nuclei and organellenucleoids was detected only in the two layers of cells at theproximal end, which is a striking characteristic of monocotyledonousplants. Moreover, to determine quantitatively the activity ofDNA synthesis in cell nuclei and organelle nucleoids in micro-scalesections of plant tissues, we developed novel techniques formicro-scale hybridization and immuno-detection analysis. Atthe distal end of the root apical meristem, DNA levels of plastidsand mitochondria were 4-fold and 5-fold greater than those inthe elongation zone, respectively. Intracellular organelle DNAlevels dropped rapidly as the distance from the root tip increased.The activity of organelle DNA synthesis in the distal end ofthe root apical meristem was about 10-fold greater than thatin the elongation zone. Our present results confirm that nuclearand organelle DNA synthesis are not synchronized, but the latteroccurs preferentially before multiple cell divisions. Key words: Organelle DNA synthesis, organelle nucleoids (organelle nuclei), root apical meristem, anti-bromo-deoxyuridine immunofluorescence microscopy, rice.