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.     

Root Hair Development

Root hairs are projections from the epidermal cells of the root that are thought to increase its effective surface area for nutrient and water uptake, enlarge the volume of exploited soil, and aid in anchoring the plant to the soil. Their formation occurs as a series of developmental processes starting with cell fate specification in the meristem. The root-hair-forming epidermal cell, or trichoblast, then participates in the diffuse growth phase associated with the elongation of the main root axis. After the fully elongated trichoblast exits the elongation zone, growth is reorganized and localized to the side in the process of root hair initiation. Initiation is then followed by a sustained phase of tip growth until the hair reaches its mature length. Thus, root hairs provide insight into a range of developmental processes from cell fate determination to growth control. The theme emerging from the molecular analysis of the control of root hair formation is that many regulators act at several stages of development. Root hair formation is also responsive to a multitude of nutrient and other environmental stimuli. Therefore, one explanation for the presence of the complex networks that regulate root hair morphogenesis may lie in the need to coordinate their highly plastic developmental program and entrain it to the current soil microenvironment being explored by the root.     

Root Hair Development

Root hairs are projections from the epidermal cells of the root that are thought to increase its effective surface area for nutrient and water uptake, enlarge the volume of exploited soil, and aid in anchoring the plant to the soil. Their formation occurs as a series of developmental processes starting with cell fate specification in the meristem. The root-hair-forming epidermal cell, or trichoblast, then participates in the diffuse growth phase associated with the elongation of the main root axis. After the fully elongated trichoblast exits the elongation zone, growth is reorganized and localized to the side in the process of root hair initiation. Initiation is then followed by a sustained phase of tip growth until the hair reaches its mature length. Thus, root hairs provide insight into a range of developmental processes from cell fate determination to growth control. The theme emerging from the molecular analysis of the control of root hair formation is that many regulators act at several stages of development. Root hair formation is also responsive to a multitude of nutrient and other environmental stimuli. Therefore, one explanation for the presence of the complex networks that regulate root hair morphogenesis may lie in the need to coordinate their highly plastic developmental program and entrain it to the current soil microenvironment being explored by the root.     

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.     

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.     

低磷供应对拟南芥根系构型的影响

在人工气候箱中,采用Johnson培养基对拟南芥在低磷供应条件下根系构型的变化进行了研究,结果表明：拟南芥在磷饥饿诱导下,主根缩短,侧根密度、根毛的数量和长度显著增加,并且,根尖到第一侧根和第一根毛的距离也大大缩短。这些改变增加了根系比表面积,并且使得根系分布更加靠近土壤表层,有利于提高植物吸收土壤中有机磷的效率。低磷胁迫还导致拟南芥根系分生组织区细胞形状变异,柱细胞数量减少;主根生长和细胞伸长的动力学分析显示,磷饥饿促使拟南芥主根生长变缓,细胞长度随磷饥饿程度的加深迅速缩小。CycB1;1:GUS染色分析结果表明,低磷破坏拟南芥根系分生组织细胞分裂能力,这些结果说明磷胁迫同时抑制了细胞的伸长和分裂,从而引起拟南芥主根的缩短。     

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.     

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.     

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.     

The Effect of Coumarin on Root Growth and Root Histology

The effect of coumarin on the root growth was studied on roots from intact plants, isolated roots and isolated elongating zones. All material was cultivated aseptically. A new method was developed for sterile culture of intact plants in flowing nutrient medium. The effects on cell division and cell elongation were studied separately. An effect on both these processes can be established at all concentrations that affect the root growth. The concentration-growth curve has an “all-or-none” appearance. Coumarin inhibits the transverse divisions in all cell layers; the perivascular layers seem to be more sensitive. Also the mitotic activity that is involved in the initiation of laterals is inhibited. The longitudinal divisions within the stele are enhanced. Coumarin decreases the cell length in all cell layers, most likely with greater relative sensitivity in the perivascular layers. Studies on the time course of cell elongation in both attached corn roots and isolated elongating zones reveal that the decrease in cell length is caused exclusively by a decrease in the maximal rate of elongation, whereas the duration of the elongation is unchanged. With each decrease of the cell length, the cell diameter is increased. The two changes are intimately connected within the greater part of the active region of concentration. Studies on the time course of the radial expansion in isolated elongating zones show a strict connection in time between cell elongation and radial expansion. The radial expansion leads to unchanged or increased cell volume at most concentrations and for most cell types. Coumarin causes an inhibition of the longitudinally directed processes and a stimulation of the radially directed ones. This is interpreted as indicating that the formative system is disengaged or reorientated, i.e., the polarity of the cells is changed. Through experiments partly with isolated elongating zones and partly by disruption of the linear phase by means of mannitol, the inhibitory effect of coumarin could be localized to the first non-linear phase of the elongation. The results were compared with earlier findings in the literature. The microtubuli are proposed as a conceivable main Component in the formative system common to both cell division and cell elongation. These are assumed to be affected by changes in the SH/SS balance produced by coumarin.     

Regulation of cell length in the Arabidopsis thaliana root by the ethylene precursor 1-aminocyclopropane- 1-carboxylic acid: a matter of apoplastic reactions

Treatment of the Arabidopsis thaliana root with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) immediately imposes a reduced maximal cell length beyond which further elongation is blocked. Here, we investigated possible apoplastic reactions involved in the inhibition of cell elongation. Five-day-old Arabidopsis seedlings were transferred to a growth medium supplemented with ACC and the effect on root cell length was recorded after 3 h of treatment. Altered characteristics in the apoplast of the nonelongating cells in the ACC-treated root, such as 'reactive oxygen species' (ROS) production and callose deposition, were detected using specific fluorochromes. The presence of functional hydroxyproline-rich glycoproteins (HRGPs) and the crosslinking of these cell-wall proteins are essential in limiting cell elongation. The ROS that drive the oxidative crosslinking of HRGPs, accumulate in the apoplast of cells in the zone where cell elongation stops. In the same cells, callose is deposited in the cell wall. The final cell length in the Arabidopsis root treated for a short period with ACC is determined in the zone of fast elongation. Both HRGPs crosslinking by ROS and callose deposition in the cell wall of this zone are suggested as causes for the reduced cell elongation.     

Tissue zinc distribution in maize seedling roots and its action on growth

Zinc (Zn) distribution over tissues and organs of maize (Zea mays L.) seedlings and its action on root growth, cell division, and cell elongation were studied. Two-day-old seedlings were incubated in the 0.25-strength Hoagland solution containing 2 or 475 μM Zn(NO₃)₂. Zn toxicity was assessed after the inhibition of primary root increment during the first and second days of incubation. The content of Zn was determined by atomic absorption spectrometry in the apical (the first centimeter from the root tip) and basal (the third centimeter from the kernel) root parts. Zn distribution in various tissues was studied by histochemical methods, using a metallochromic indicator zincon and fluorescent indicator Zinpyr-1 and light and confocal scanning fluorescent light microscopy, respectively. To evaluate Zn effects on growth processes, the average length of the meristem; the length of fully elongated cells; the number of meristematic cells in the cortex row; and duration of the cell cycle were measured. When the Zn concentration in the solution was high, the Zn content per weight unit was higher in the basal root part due to its accumulation in lateral root primordial. Zn was also accumulated in both the meristem apoplast and cell protoplasts. In the basal and middle root parts, Zn was detected essentially in all tissues predominantly in the apoplast. Zn inhibited both cell division and elongation. Under Zn influence, the size of the meristem and the number of meristematic cells decreased, which was determined by an increase in the cell cycle duration. The length of the fully elongated cells was also reduced. A comparison of Zn distribution and growth-suppressing activity with other heavy metals studied earlier allows a conclusion that toxic action of heavy metals is mainly determined by physical and chemical properties of their ions and specific patterns of their transport and distribution. As a result, two basic processes determining root growth, e.g., cell division and elongation, could be affected differently.     

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.     

Does salinity reduce growth in maize root epidermal cells by inhibiting their capacity for cell wall acidification?

The reduction in growth of maize (Zea mays L.) seedling primary roots induced by salinization of the nutrient medium with 100 millimolar NaCl was accompanied by reductions in the length of the root tip elongation zone, the length of fully elongated epidermal cells, and the apparent rate of cell production: Each was partially restored when calcium levels in the salinized growth medium were increased from 0.5 to 10.0 millimolar. We investigated the possibility that the inhibition of elongation growth by salinity might be associated with an inhibition of cell wall acidification, such as that which occurs when root growth is inhibited by IAA. A qualitative assay of root surface acidification, using bromocresol purple pH indicator in agar, showed that salinized roots, with and without extra calcium, produced a zone of surface acidification which was similar to that produced by control roots. The zone of acidification began 1 to 2 millimeters behind the tip and coincided with the zone of cell elongation. The remainder of the root alkalinized its surface. Kinetics of surface acidification were assayed quantitatively by placing a flat tipped pH electrode in contact with the elongation zone. The pH at the epidermal surfaces of roots grown either with 100 millimolar NaCl (growth inhibitory), or with 10 millimolar calcium ± NaCl (little growth inhibition), declined from 6.0 to 5.1 over 30 minutes. We conclude that NaCl did not inhibit growth by reducing the capacity of epidermal cells to acidify their walls.     

Shade avoidance responses are mediated by the ATHB-2 HD-zip protein, a negative regulator of gene expression.

The ATHB-2 gene encoding an homeodomain-leucine zipper protein is rapidly and strongly induced by changes in the ratio of red to far-red light which naturally occur during the daytime under the canopy and induce in many plants the shade avoidance response. Here, we show that elevated ATHB-2 levels inhibit cotyledon expansion by restricting cell elongation in the cotyledon-length and -width direction. We also show that elevated ATHB-2 levels enhance longitudinal cell expansion in the hypocotyl. Interestingly, we found that ATHB-2-induced, as well as shade-induced, elongation of the hypocotyl is dependent on the auxin transport system. In the root and hypocotyl, elevated ATHB-2 levels also inhibit specific cell proliferation such as secondary growth of the vascular system and lateral root formation. Consistent with the key role of auxin in these processes, we found that auxin is able to rescue the ATHB-2 lateral root phenotype. We also show that reduced levels of ATHB-2 result in reciprocal phenotypes. Moreover, we demonstrate that ATHB-2 functions as a negative regulator of gene expression in a transient assay. Remarkably, the expression in transgenic plants of a derivative of ATHB-2 with the same DNA binding specificity but opposite regulatory properties results in a shift in the orientation of hypocotyl cell expansion toward radial expansion, and in an increase in hypocotyl secondary cell proliferation. A model of ATHB-2 function in the regulation of shade-induced growth responses is proposed.     

Phosphate starvation induces a determinate developmental program in the roots of Arabidopsis thaliana

When growing under limiting phosphate (P) conditions, Arabidopsis thaliana plants show dramatic changes in root architecture, including a reduction in primary root length, increased formation of lateral roots and greater formation of root hairs. Here we report that primary root growth inhibition by low P is caused by a shift from an indeterminate to a determinate developmental program. In the primary root, the low P-induced determinate growth program initiates with a reduction of cell elongation followed by the progressive loss of meristematic cells. At later stages, cell proliferation ceases and cell differentiation takes place at the former cell elongation and meristematic regions of the primary root. In low P, not only the primary but also almost all mature lateral roots enter the determinate developmental program. Kinetic studies of expression of the cell cycle marker CycB1;1:uidA and the quiescent center (QC) identity marker QC46:GUS showed that in low P conditions, reduction in proliferation in the primary root was preceded by alterations in the QC. These results suggest that in Arabidopsis, P limitation can induce a determinate root developmental program that plays an important role in altering root system architecture and that the QC could act as a sensor of environmental signals.     

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.     

The Petunia tra1 gene controls cell elongation and plant development, and mediates responses to cytokinins

The molecular control of cell elongation, one of the basic processes of plant morphogenesis, is still largely not understood. This paper describes a Petunia hybrida mutant of dumpy phenotype, trapu, which identifies tra1, a gene required for cell elongation and mediating responses to applied cytokinin. This mutant displayed an extreme reduction in length, due to a single recessive mutation which was expressed in every part of the plant and during the entire life of the plant, including the mature embryo. The mutant was unable to flower. The mutant roots, as well as the leafy organs, were short and thick, and the root elongation zone, hypocotyl and petioles were absent. The mutant plantlets responded neither to applied auxin nor to gibberellin, indicating that this phenotype was not caused by a deprivation of these phytohormones. However, unlike the wildtype, the mutant growth was stimulated by applied cytokinin, even though its morphology remained abnormal. A histological study revealed the presence of all tissue types in normal positions, including root hairs and vascular bundles. The mutant's cells were rounder in every tissue. Both shoot and root meristems were disorganized, without consistent cell shape and size. The regular cell files, which are typical of a normal root apex organization, were totally absent in the mutant root apex. Indirect immunofluorescence of α-tubulin on root apices showed the cortical microtubules in the mutant cells to be unable to form the parallel arrays in elongating cells and the preprophase band in dividing cells. This default resulted in the prevention of unidirectional cell elongation and formation of regular cell files, thus causing the trapu phenotype. This paper discusses the similarities and differences of trapu to the Arabidopsis mutants, fass and ton, trapu confirming that the establishment of plant body pattern and differentiation can be dissociated from cell elongation.     

Gibberellic acid and dwarfism effects on the growth dynamics of B73 maize (Zea mays L.) leaf blades: a transient increase in apoplastic peroxidase activity precedes cessation of cell elongation

The relationship between apoplastic peroxidase (EC 1.11.1.7) activity and cessation of growth in maize (Zea mays L.) leaf blades was investigated by altering elongation zone length. Apoplastic peroxidase activity in the elongation and secondary cell wall deposition zones of elongating leaf blades of the maize inbred line B73 was used as a control and compared to leaves of the dwarf mutant D8-81127, a near-isogenic line of B73 unresponsive to gibberellins, and to leaves of B73 plants to which gibberellic acid (GA(3)) had been applied via root uptake. Elongation zone length was increased by treatment with GA(3) through an increase in cell number as well as increased final cell length. The shorter elongation zone of dwarf leaves occurred primarily through reduced final cell length. Although elongation zone length differed among dwarf, control, and GA(3)-treated leaf blades, in all three treatments a transient increase in apoplastic peroxidase activity preceded a reduction in the segmental elongation rate in leaves. A peroxidase isoenzyme with pI 7.0 occurred in the leaf elongation zone during growth deceleration in all three treatments, and its activity decreased as growth displaced tissue into the region of secondary cell wall deposition. Growth cessation for all treatments coincided with the first appearance of peroxidase isozymes with pIs of 5.6 and 5.7. Based on the activity of particular isozymes relative to growth and differentiation, the pI 7.0 isoenzyme is most likely to be involved in cessation of cell elongation, while isozymes with pIs 5.6 and 5.7 are likely to be active in lignification.