The Time Effect of Auxin and Calcium on Growth and Elastic Modulus in Hypocotyls

The relationship between Young's modulus and longitudinal growth has been studied on growing segments of etiolated sunflower hypocotyls. The modulus was determined by means of the resonance frequency method. IAA in a concentration of 2.8 10^?5 M produces a decrease in the modulus with a time lag of 4 minutes, while an increase in growth is observable only after 6 minutes. Addition of IAA results in a stronger decrease in the modulus if the segments are placed in a solution of 0.1 M mannitol rattier Hum in water. Through plasmometric measurements it has been found that the elastic extensibility is insignificant compared with the growth. After the addition of IAA, there occurs a marked elongation both in 0.1 M mannitol and in water, and at the same time a decrease in the elastic extensibility of the segments is observed. In the growing segments an increased modulus causes an in creased elastic extensibility, a matter which is directly contrary to the relationship found in a physical system with an applied external force. An explanation of this discrepancy has been given. With an excess of calcium the modulus increases, while the elongation decreases. Calcium deficiency implies both a decreased modulus and a decreased growth. With the addition of 10^?3 M Ca(NO₃)₂ to segments raised without calcium the modulus increases after only 10 minutes, while an increase in longitudinal elongation is observable after 30 minutes. With the addition of IAA to the calcium deficient segments the modulus decreases to the same extent as in an optimal supply o f calcium. The results are discussed with reference to other investigations about elasticity and growth. It has been concluded that plastic extensibility cannot he of great importance in longitudinal growth. Time studies of the auxin effect and I he interaction between auxin and calcium have confirmed the hypothesis that one of the primary actions of auxin consists in a loosening of the cell wall matrix. Calcium always increases Youngs modulus and gives the cell wall a more rigid structure. Furthermore, calcium in a certain concentration is necessary for longitudinal growth.     

A Fine Structural Analysis of Auxin-induced Elongation of Cucumber Hypocotyls, and the Effects of Calcium Antagonists and Ionophores

The fine structure of epidermal cells, particularly in relationto dictyosomes, has been examined in different regions of dark-growncucumber hypocotyls and in response to auxin treatment, usingboth dot overlay and image analysis techniques. The most noticeablechange in cell structure along the hypocotyls is the increasein vacuolar volume. The volume fraction occupied by dictyosomesand secretory vesicles also increased, whereas that for mitochondriaremained relatively constant. During auxin treatment, the volumefraction for dictyosomes showed an increase after 30 min followedby a fall, whereas that occupied by secretory vesicles fellsteadily over 90 min. The number of cisternae per dictyosomeshowed some increase after 2 h of auxin treatment, althoughthe increase in dictyosomal material with cell expansion waslargely accounted for by an increase in the number of dictyosomes. Auxin-stimulated elongation growth of the hypocotyls was inhibitedby a range of calcium antagonists, chelators and ionophores.The most marked inhibitions were observed with calcium chloride,the chelator chlortetracycline and the ionophores verapamil,nigericin and monensin. Linear transducer experiments showedthat these compounds generally caused an immediate reductionin the rate of growth. Fine structural observations carriedout on epidermal cells showed the most obvious effects withmonensin and nigericin which caused dictyosomes and secretoryvesicles to swell. EGTA and LaCl₃ caused secretory vesiclesto accumulate around dictyosomes, while the ionophore A23187had little effect. The results suggest that the concentration of Ca²⁺ in the cytoplasmmay be critical for cell elongation. Compounds which chelateCa²⁺ appear to be more effective inhibitors of growth in theinitial acid-induced phase, whereas those which affect ionicgradients are more disruptive in the second phase.Copyright1993, 1999 Academic Press Calcium, Cucumis sativus hypocotyle, dictyosomes, elongation growth, indoleacetic acid, stereology     

Proton efflux from oat coleoptile cells and exchange with wall calcium after IAA or fusicoccin treatment

Elongation growth of plant cells occurs by stretching of cell walls under turgor pressure when intermolecular bonds in the walls are temporarily loosened. The acid-growth theory predicts that wall loosening is the result of wall acidification because treatments (including IAA and fusicoccin) that cause lowered wall pH cause elongation. However, conclusive evidence that IAA primarily reduces wall pH has been lacking. Calcium has been reported to stiffen the cell walls. We have used a microelectrode ion-flux measuring technique to observe directly, and non-invasively, the net fluxes of protons and calcium from split coleoptiles of oats (Avena sativa L.) in unbuffered solution. Normal net fluxes are 10 nmol · m⁻² · s⁻¹ proton efflux and zero calcium flux. The toxin fusicoccin (1 μM) causes immediate efflux from tissue not only of protons, but also of calcium, about 110 nmol · m⁻² · s⁻¹ in each case. The data fit the “weak acid Donnan Manning” model for ion exchange in the cell wall. Thus we associate the known “acid-growth” effect of fusicoccin with the displacement of calcium from the wall by exchange for protons extruded from the cytoplasm. Application of 10 μM IAA causes proton efflux to increase transiently by about 15 nmol · m⁻² · s⁻¹ with a lag of about 10 min. The calcium influx decreases immediately to an efflux of about 20 nmol · m⁻² · s⁻¹. It appears that auxin too causes an “acid-growth” effect, with extruded protons exchanging for calcium in the cell walls. I. Arif is currently recieving an AIDAB scholarship. This work was supported by an Australian Research Council grant to I.A. Newman.     

Role of the plasma membrane H<Superscript>+</Superscript>-ATPase in auxin-induced elongation growth: historical and new aspects

This article will cover historical and recent aspects of reactions and mechanisms involved in the auxin-induced signalling cascade that terminates in the dramatic elongation growth of cells and plant organs. Massive evidence has accumulated that the final target of auxin action is the plasma membrane H⁺-ATPase, which excretes H⁺ ions into the cell wall compartment and, in an antiport, takes up K⁺ ions through an inwardly rectifying K⁺ channel. The auxin-enhanced H⁺ pumping lowers the cell wall pH, activates pH-sensitive enzymes and proteins within the wall, and initiates cell-wall loosening and extension growth. These processes, induced by auxin or by the "super-auxin" fusicoccin, can be blocked instantly and specifically by a voltage inhibition of the H⁺-ATPase due to removal of K⁺ ions or the addition of K⁺-channel blockers. Vice versa, H⁺ pumping and growth are immediately switched on by addition of K⁺ ions. Furthermore, the treatment of segments either with auxin or with fusicoccin (which activates the H⁺-ATPase irreversibly) or with acid buffers (from outside) causes an identical transformation and degradation pattern of cell wall constituents during cell-wall loosening and growth. These and other results described below are in agreement with the acid-growth theory of elongation growth. However, objections to this theory are also discussed.     

The Effect of Calcium Antagonists on Auxin-induced Elongation and on the Expression of Two Auxin-Regulated Genes in Pea Epicotyls

Calcium has been implicated in various regulatory roles in plantcells including auxin-induced cell elongation. Treatment ofpea epicotyl segments with the calcium chelators, EGTA and chlorotetracycline(CTC), the calcium ionophore, A23187 [GenBank] , and channel blocker, D-600,inhibits auxin-induced cell elongation. Depletion of tissuecalcium either by EGTA or EGTA and a calcium ionophore doesnot interfere with the induction of the early auxin induciblemRNAs pIAA4/5 and pIAA6. Similarly, an increase in cytosoliccalcium with calcium and calcium ionophore neither induces thehormonally regulated mRNAs nor interferes with their inductionby auxin. The calcium channel blocker, D-600, is without effecton the auxin-regulated mRNA induction. The results indicatethat calcium is not involved in the rapid induction of IAA4/5and IAA6 genes in pea tissue. However, a possible role for calciumin the translation of these mRNAs, or in the expression of otherauxin-regulated genes, is not excluded. ³Present Address: Department of Biology, Tokyo MetropolitanUniversity, Tokyo, Japan. (Received April 8, 1988; Accepted July 30, 1988)     

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.     

INFLUENCE OF THE TONIC EFFECT OF GRAVITATION AND AUXIN ON CELL ELONGATION AND POLARITY IN ROOTS

The influence of a longitudinal (tonic) gravitational force and of auxin on the pattern of growth and cell polarity has been studied on intact roots of wheat seedlings. A klinostat technique was used for controlling gravitation. Growth in length was evaluated as cell division activity, rate of cell elongation (μ/h) and duration of elongation (h). Exogenous auxin (1-NAA) increases the rate of cell elongation in all concentrations tested (10⁻⁸ — 3 × 10⁻⁷m ) and shortens the time of elongation with increasing concentration. It promotes rate of cell elongation in roots as it does in shoots. It also accentuates the polar insertion of root hairs and their growth. The tonic effect of gravitation resembles that of an increase in auxin both in light and darkness. The results are discussed in relation to plagiotropic growth of roots, root growth promotions by auxin, and the difference between root and shoot growth.     

Root gravitropism

When a plant root is reoriented within the gravity field, it responds by initiating a curvature which eventually results in vertical growth. Gravity sensing occurs primarily in the root tip. It may involve amyloplast sedimentation in the columella cells of the root cap, or the detection of forces exerted by the mass of the protoplast on opposite sides of its cell wall. Gravisensing activates a signal transduction cascade which results in the asymmetric redistribution of auxin and apoplastic Ca²⁺ across the root tip, with accumulation at the bottom side. The resulting lateral asymmetry in Ca²⁺ and auxin concentration is probably transmitted to the elongation zone where differential cellular elongation occurs until the tip resumes vertical growth. The Cholodny-Went theory proposes that gravity-induced auxin redistribution across a gravistimulated plant organ is responsible for the gravitropic response. However, recent data indicate that the gravity-induced reorientation is more complex, involving both auxin gradient-dependent and auxin gradient-independent events.     

Relationship between Promotion of Xyloglucan Metabolism and Induction of Elongation by Indoleacetic Acid

Auxin promotes the liberation of a xlyoglucan polymer from the cell walls of elongating pea (Pisum sativum) stem segments. The released polymer can be isolated from the polysaccharide fraction of the water-soluble portion of tissue homogenates, thus providing as assay for this kind of metabolism. Promotion of xyloglucan metabolism by auxin begins within 15 minutes of hormone presentation. The effect increases with auxin concentration in a manner similar to the hormone effect on elongation. However, the xyloglucan effect of auxin occurs perfectly normally when elongation is completely blocked by mannitol. Metabolic inhibitors and Ca²⁺, on the other hand, inhibit auxin promotion of elongation and of xyloglucan metabolism in parallel. The results suggest that the changes in xyloglucan reflect the means by which auxin modifies the cell wall to cause elongation.     

Reorientation of Microfibrils and Microtubules at the Outer Epidermal Wall of Maize Coleoptiles During Auxin-Mediated Growth

Auxin-mediated elongation growth of isolated subapical coleoptile segments of maize (Zea mays L.) is controlled by the extensibility of the outer cell wall of the outer epidermis (Kutschera et al., 1987). Here we investigate the hypothesis that auxin controls the extensibility of this wall by changing the orientation of newly deposited microfibrils through a corresponding change in the orientation of cortical microtubules. On the basis of electron micrographs it is shown that cessation of growth after removal of the endogenous source of auxin is correlated with a relative increase of longitudinally orientated microfibrils and microtubules at the inner wall surface. Conversely, reinduction of growth by exogenous auxin is correlated with a relative increase of transversely orientated microfibrils and microtubules at the inner wall surface. These changes can be detected 30–60 min after the removal and addition of auxin, respectively. The functional significance of directional changes of newly desposited wall microfibrils for the control of elongation growth is discussed.     

CELL WALL SYNTHESIS AND CELL ELONGATION IN OAT COLEOPTILE TISSUE

Ray , Peter M. (U. Michigan, Ann Arbor.) Cell wall synthesis and cell elongation in oat coleoptile tissue. Amer. Jour. Bot. 49(9): 928–939. Illus. 1962.—Cell wall synthesis in oat coleoptile cylinders tends to run parallel with but not usually proportional to cell elongation both under promotion by auxin and sugar and under inhibition by supraoptimal auxin or sugar, or by a variety of other inhibitors. Inhibitors of elongation fall into 2 classes with respect to their effects on wall synthesis: (1) those which inhibit the 2 processes approximately equally (galactose, mannose, mannitol, azide, iodoacetate, dinitrophenol, low temperature, supraoptimal auxin) and (2) those which inhibit elongation percentagewise much more strongly than wall synthesis, so that as complete inhibition of elongation is approached, substantial wall synthesis continues (Ca^{+ +}, fluoride, arsenite, mercurials). When coleoptile cylinders elongate in the absence of sugar, the cell walls appear to become markedly thinner, and in some experiments negligible increase in total wall material apparently occurs. However, the amount of α-cellulose does rise. Increase in cell wall material occurs during elongation of cylinders at 2 C. The results are interpreted as indicating that during elongation the bulk of new cell wall material is added by apposition, but a certain proportion of the new material is probably introduced within the existing wall structure and induces its expansion.     

Rapid cellular responses to auxin and the regulation of growth

Abstract The cellular responses rapidly evoked by auxin are reviewed, and related to a consideration of how growth rate is regulated in excised segments and in whole dicotyledonous plants. Two processes, synthesis of proteins and of cell wall components, are both promoted by auxin and essential for auxin-stimulated growth, whereas other processes show little promotion by auxin or do not appear essential for growth. Current models for the cellular regulation of growth by auxin are briefly discussed, and a new model presented. Auxin is suggested to act by bringing about a transient increase in cytosolic Ca²⁺ levels, which through the stimulation of protein kinases converts a cytoplasmic protein factor to an active state capable of binding auxin. The protein-auxin complex induces mRNA synthesis, which effects the increased synthesis of cell wall components and their incorporation into the wall, resulting in wall loosening and growth. It is proposed that the factor limiting growth in floating excised segments may initially be cell wall pH, but that this is not the case in whole plants and growth is instead mediated by increased protein and matrix cell wall synthesis. Differences are noted between monocotyledonous coleoptiles and dicotyledonous stems in some metabolic processes possibly involved in auxin growth responses, and it is cautioned that observations made on one tissue may not necessarily be applicable to the other. Care should also be taken in applying conclusions drawn from studies on excised tissue to the interpretation of growth regulation in the whole plant.     

The effect of ethylene and auxin on cell wall extensibility of the Semi-aquatic fern,Regnellidium diphyllum

Ethylene and auxin both enhance cell elongation growth in the rachis of the frond of Regnellidium diphyllum. Measurements of the stress relaxation modulus of the walls of methanol-killed rachis segments show that both auxin and ethylene cause an increase in cell wall extensibility, that the effects are additive, and that they occur in the presence of hypertonic solutions of mannitol that preclude cell elongation. The results are taken as evidence for the operation of two separate mechanisms for cell wall loosening.Abbreviation IAA indol-3yl-acetic acid     

Reevaluation of the Effect of Calcium Ions on Auxin-induced Elongation

The mechanism by which calcium ions inhibit cell elongation has been reinvestigated. Growth-inhibiting levels of calcium, when applied to isolated walls (in vitro treatment) do not decrease cell wall extensibility as measured by the Instron technique. Thus, the hypothesis that calcium inhibits growth by forming wall-stiffening calcium bridges must be abandoned. Treatment of living auxin-treated sections with calcium (in vivo treatment) does cause a decrease in the subsequently measured wall extensibility, but this decline appears to be simply a consequence of the growth inhibition rather than its cause. Growth-inhibiting levels of calcium do not appreciably reduce the rate of auxin-enhanced H(+) excretion. Pretreatment with calcium does not reduce the capacity of walls to undergo acid-activated wall loosening in the absence of calcium. High concentrations of CaCl(2) (0.02 m) cause an initial elastic shrinkage of Avena sections comparable to that caused by the same osmolarity of mannitol, but the subsequent growth inhibition is too great to be explained by an osmotic inhibition. Calcium ions do inhibit H(+)-induced extension of frozen-thawed sections under tension. The growth-inhibitory effects of calcium, then, may be ascribed to a direct inhibition exerted by calcium ions on the H(+)-induced wall-loosening process.     

Galactose inhibition of auxin-induced cell elongation in oat coleoptile segments

Auxin-induced cell elongation in oat coleoptile segments was inhibited by galactose; removal of galactose restored growth. Galactose did not appear to affect the following factors which modify cell elongation: auxin uptake, auxin metabolism, osmotic concentration of cell sap, uptake of tritium-labeled water, auxin-induced wall loosening as measured by a decrease in the minimum stress-relaxation time and auxininduced glucan degradation. Galactose markedly prevented incorporation of [¹⁴C]-glucose into cellulosic and non-cellulosic fractions of the cell wall. It was concluded that galactose inhibited auxin-induced long-term elongation of oat coleoptile segments by interfering with cell wall synthesis.     

Promotion of Xyloglucan Metabolism by Acid pH

Like indoleacetic acid, buffers of acidic pH, which stimulate elongation of pea (Pisum sativum var. Alaska) stem tissue, induce the appearance within the tissue of a watersoluble xyloglucan polymer that probably arises from previously deposited wall material. Neutral pH buffers, which inhibit the elongation response to indoleacetic acid in this tissue, inhibit indoleacetic acid-induced increase in soluble xyloglucan. The findings provide further evidence that release of soluble xyloglucan from the cell walls of pea results from the biochemical action on the cell wall that is responsible for wall extension. The data also indicate that treatment of tissue with either auxin or acidic pH has a similar biochemical effect on the cell wall. This is consistent with the H⁺ secretion theory of auxin action.     

Ion channels meet auxin action

The regulation of cell division and elongation in plants is accomplished by the action of different phytohormones. Auxin as one of these growth regulators is known to stimulate cell elongation growth in the aerial parts of the plant. Here, auxin enhances cell enlargement by increasing the extensibility of the cell wall and by facilitating the uptake of osmolytes such as potassium ions into the cell. Starting in the late 1990s, the auxin regulation of ion channels mediating K+ import into the cell has been studied in great detail. In this article we will focus on the molecular mechanisms underlying the modulation of K+ transport by auxin and present a model to explain how the regulation of K+ channels is involved in auxin-induced cell elongation growth.     

Auxin regulation and spatial localization of an endo-1,4-β-D-glucanase and a xyloglucan endotransglycosylase in expanding tomato hypocotyls

Xyloglucan, the primary hemicellulosic cell wall polysaccharide in dicotyledons, undergoes substantial modification during auxin-stimulated cell expansion. To identify candidates for mediating xyloglucan turnover, the expression and auxin regulation of tomato Cel7 and LeEXT , genes encoding an endo-1,4-β-glucanase (EGase) and a xyloglucan endotransglycosylase (XET), respectively, were examined. LeEXT mRNA was present primarily in elongating regions of the hypocotyl and was induced to higher levels by hormone treatments that elicited elongation of hypocotyl segments. Cel7 mRNA abundance was very low in both elongating and mature regions of the hypocotyl but was induced to accumulate to high levels in both hypocotyl regions by auxin application. Analysis of the time dependence of expression of Cel7 and LeEXT during auxin treatment suggested that induction of these genes is not required for rapid growth responses but may participate in the cell wall changes involved in sustained cell elongation. Localization of Cel7 and LeEXT mRNA by in situ hybridization revealed that both genes are expressed in outer cell layers of the hypocotyl. In untreated etiolated seedlings, LeEXT mRNA was detected in epidermal cells of the elongating region, a tissue considered to play a key role in auxin-induced elongation. After auxin treatment, Cel7 and LeEXT mRNA showed an overlapping spatial distribution in the epidermis and outer cortical cell layers. We conclude that LeEXT and Cel7 exhibit both unique and overlapping patterns of expression and have the potential to act cooperatively in mediating cell wall disassembly associated with expansive growth.     

Influence of Auxin on Young's Modulus in Stems and Roots of Pisum and the Theory of Changing the Modulus in Tissues

The effect of auxin on elastic extensibility has been investigated by means of the resonance frequency melhod in Pisum, sativum. The time lag for the decrease in Young's modulus E, caused by IAA, was between 2 and 3 minutes in etiolated stem internodes. The time lag for growth was about 7 minutes. The measurements of E in root segments were only qualitative owing to the structural characteristics; IAA decreases E in roots as it does in stems, but only in the region where IAA is assumed to enhance elongation. The connexion between elastic modulus and growth is discussed with reference to other investigations. The assumption has been made that a decrease in elastic modulus indicates a change in the cell wall which in some way is conducive to growth (induction of elongation). The theoretical possibilities of changing E have been discussed with reference to the formula for water fluxes. Both a change in a cell wall properly and a change in the cytoplasmic permeability are able to cause a change in E in the same way as auxin does. An early action of auxin must be located in the cell-wall-plasmalemma region.     

WEG1, which encodes a cell wall hydroxyproline-rich glycoprotein,is essential for parental root elongation controlling lateral root formation in rice

Lateral roots (LRs) determine the overall root system architecture, thus enabling plants to efficiently explore their underground environment for water and nutrients. However, the mechanisms regulating LR development are poorly understood in monocotyledonous plants. We characterized a rice mutant, wavy root elongation growth 1 (weg1), that produced higher number of long and thick LRs (L-type LRs) formed from the curvatures of its wavy parental roots caused by asymmetric cell growth in the elongation zone. Consistent with this phenotype, was the expression of the WEG1 gene, which encodes a putative member of the hydroxyproline-rich glycoprotein family that regulates cell wall extensibility, in the root elongation zone. The asymmetric elongation growth in roots is well known to be regulated by auxin, but we found that the distribution of auxin at the apical region of the mutant and the wild-type roots was symmetric suggesting that the wavy root phenotype in rice is independent of auxin. However, the accumulation of auxin at the convex side of the curvatures, the site of L-type LR formation, suggested that auxin likely induced the formation of L-type LRs. This was supported by the need of a high amount of exogenous auxin to induce the formation of L-type LRs. These results suggest that the MNU-induced weg1 mutated gene regulates the auxin-independent parental root elongation that controls the number of likely auxin-induced L-type LRs, thus reflecting its importance in improving rice root architecture.