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.     

Stimulation of Pisum sativum epicotyl elongation by gibberellin and auxin. – Different effects of two hormones on osmoregulation and cell walls

The possible involvement of auxin in the action of gibberellin in stimulating cell elongation was examined by comparing the effects of gibberellic acid (GA) and IAA on the growth, osmoregulation and cell wall properties of the Alaska pea ( Pisum sativum L. cv. Alaska) subhook. Both GA and IAA stimulated cell elongation in the subhook region of derooted cuttings. Cotyledon excision decreased the stimulating effect of GA on the growth of the subhook region, but did not affect that of IAA. As the subhook region elongated, the osmotic potential of the cell sap and the total amount of osmotic solutes increased. Cotyledon excision accelerated the increase in the osmotic potential and suppressed the accumulation of osmotic solutes. In cuttings with cotyledons. GA partly counteracted the increase in the osmotic potential and substantially promoted the accumulation of osmotic solutes. On the other hand, in cuttings without cotyledons. GA did not affect the change in the osmotic potential although it slightly promoted the accumulation of osmotic solutes. IAA accelerated the increase in the osmotic potential, but did not affect the accumulation of osmotic solutes. IAA enhanced the extensibility of the cell wall, while GA did not affect it. These results suggest that at least in the Alaksa pea subhook region. GA does not stimulate cell elongation by affecting the level of auxin.     

Genetic Dissection of the Relative Roles of Auxin and Gibberellin in the Regulation of Stem Elongation in Intact Light-Grown Peas

Exogenous gibberellin (GA) and auxin (indoleacetic acid [IAA]) strongly stimulated stem elongation in dwarf GA1-deficient le mutants of light-grown pea (Pisum sativum L.): IAA elicited a sharp increase in growth rate after 20 min followed by a slow decline; the GA response had a longer lag (3 h) and growth increased gradually with time. These responses were additive. The effect of GA was mainly in internodes less than 25% expanded, whereas that of IAA was in the older, elongating internodes. IAA stimulated growth by cell extension; GA stimulated growth by an increase in cell length and cell number. Dwarf lkb GA-response-mutant plants elongated poorly in response to GA (accounted for by an increase in cell number) but were very responsive to IAA. GA produced a substantial elongation in lkb plants only in the presence of IAA. Because lkb plants contain low levels of IAA, growth suppression in dwarf lkb mutants seems to be due to a deficiency in endogenous auxin. GA may enhance the auxin induction of cell elongation but cannot promote elongation in the absence of auxin. The effect of GA may, in part, be mediated by auxin. Auxin and GA control separate processes that together contribute to stem elongation. A deficiency in either leads to a dwarfed phenotype.     

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     

Red Light-Regulated Growth : I. Changes in the Abundance of Indoleacetic Acid and a 22-Kilodalton Auxin-Binding Protein in the Maize Mesocotyl

We examined the changes in the levels of indoleacetic acid (IAA), IAA esters, and a 22-kilodalton subunit auxin-binding protein (ABP1) in apical mesocotyl tissue of maize (Zea mays L.) during continuous red light (R) irradiation. These changes were compared with the kinetics of R-induced growth inhibition in the same tissue. Upon the onset of continuous irradiation, growth decreased in a continuous manner following a brief lag period. The decrease in growth continued for 5 hours, then remained constant at 25% of the dark rate. The abundance of ABP1 and the level of free IAA both decreased in the mesocotyl. Only the kinetics of the decrease in IAA within the apical mesocotyl correlated with the initial change in growth, although growth continued to decrease even after IAA content reached its final level, 50% of the dark control. This decrease in IAA within the mesocotyl probably occurs primarily by a change in its transport within the shoot since auxin applied as a pulse moved basipetally in R-irradiated tissue at the same rate but with half the area as dark control tissue. In situ localization of auxin in etiolated maize shoots revealed that R-irradiated shoots contained less auxin in the epidermis than the dark controls. Irradiated mesocotyl grew 50% less than the dark controls even when incubated in an optimal level of auxin. However, irradiated and dark tissue contained essentially the same amount of radioactivity after incubation in [¹⁴C]IAA indicating that the light treatment does not affect the uptake into the tissue through the cut end, although it is possible that a small subset of cells within the mesocotyl is affected. These observations support the hypothesis that R causes a decrease in the level of auxin in epidermal cells of the mesocotyl, consequently constraining the growth of the entire mesocotyl.     

Growth, Turgor, Water Potential, and Young's Modulus in Pea Internodes

The relations between longitudinal growth, Young's modulus, turgor, water potential, and tissue tensions have been studied on growing internodes of etiolated pea seedlings in an attempt to apply some physical concepts to the growth of a well-known plant material. The modulus has been determined by the resonance frequency method and expressed as E_tissue It increases nearly proportional to the turgor pressure and is at water saturation more than 50 times higher than at plasmolysis. E_tissue is higher in the epidermis than in the ground parenchyma. Indoleacetic acid causes a decrease in Etissue Other properties have been studied on intact and split segments of internodes in solutions of graded mannitol additions. — The following tentative picture of the normal course of the growth has been obtained. Auxin induces growth both in the periphery (epidermis) and in the central core (parenchyma) under a decrease in E_tissue This is followed by an increase of E_tissue which is independent of auxin but depending upon the turgor pressure. It is assumed to involve internal structural changes of the cell walls of the type of creep. The rapid growth takes place in a dynamic system with a low water potential despite favourable water conditions. Epidermis and parenchyma grow equally rapid without tissue tensions. — Such can be produced artificially by splitting of segments and water uptake. The parenchyma thereby loses its sensitivity to auxin. This is the background of the split stem test for auxin. — E_tissue increases when growth is slowing down, probably owing to both synthesis of wall substance and structural changes within the wall. The cells attain a more static condition with E_tissue higher in epidermis than in parenchyma. This leads to the normal tissue tensions. — The result agrees with growth according to the multi-net-principle. The cause of the low water potential and low turgor is discussed with reference to the dynamic nature of both growth and water transport and a probably low matric potential of the streaming water. The decrease in E_tissue following auxin addition is small but is the net difference between an auxin-induced decrease and an increase through the assumed creep.     

Auxin Enhancement of mRNAs in Epidermis and Internal Tissues of the Pea Stem and Its Significance for Control of Elongation

The epidermis has been considered the site of auxin action on elongation of stems and coleoptiles. To try to identify mRNAs that might mediate auxin stimulation of cell enlargement, we compared, using in vitro translation assays, mRNA enhancement by indoleacetic acid (IAA) in the epidermis, with that in the internal tissues, of pea (Pisum sativum L., cv Alaska) third internode segments. We used seedlings that had been grown under red light, which enables the epidermis to be peeled efficiently from the internode. Most of the `early' IAA enhancements previously reported using etiolated peas, plus several hitherto undescribed enhancements, occur in both the epidermis and the internal tissue of the light-grown plants after 4 hours of IAA treatment. These enhancements, therefore, do not fulfill the expectation of elongation-specific mRNAs localized to the epidermis. One epidermis-specific IAA enhancement does occur, but begins only subsequent to 1 hour (but before 4 hours) of auxin treatment. Similarly, the previously mentioned IAA enhancements common to epidermis and internal tissue do not begin, in the light-grown plants, within 1 hour of IAA treatment. Since IAA stimulates elongation in light-grown internodes within 15 minutes, it appears that none of these mRNAs can be responsible for auxin induction of elongation. We confirmed, with our methods, the previous reports that some of these mRNAs are enhanced by IAA within 0.5 hour in etiolated internodes. This indicates that we could have detected an early enhancement in light-grown tissue had it occurred.     

Changes in the concentration of indole-3-acetic acid during the growth of etiolated lupin hypocotyls

The longitudinal distribution of unaltered radioactive indole-3-acetic acid (IAA), after application of [5-³H]-IAA to decapitated etiolated lupin hypocotyls. exhibited a wave-like pattern similar to that obtained with endogenous IAA. Waves of radioactive IAA were localizated both in the elongation zone and in the non-growing basal region of the hypocotyl. These IAA waves were transient because of basipetal polar transport and metabolism of IAA.
The level of endogenous IAA in different zones of the hypocotyl varied with age, following a wave-like pattern. During the elongation period of each zone, IAA was parallel to the bell-shaped curve of the growth rate. In addition, a role in secondary cell wall deposition is suggested for the other IAA wave that appeared after the cell elongation period, since an electron microscopic morphometric analysis of the cell wall showed that the cell wall thickness increased once the cell elongation ceased.
As the oscillation of endogenous IAA level occured in both space (distribution along the hypocotyl) and time (variation with age), it is suggested that the level of IAA really depended on the growth status of the cells. The response of the cells to the positional information submitted by the auxin waves as regards the growth status of the cell is discussed.     

Growth and anthocyanin synthesis in excised Sorghum internodes

Summary Ligh-induced anthocyanin synthesis in excised dark-grown internodes of Sorghum was depressed by the addition of auxin to the incubating medium at physiological concentrations. Both IAA and the synthetic auxin, 2,4-D, reduced anthocyanin yield. Similar results were obtained with isolated internode segments and in internodes incubated with coleoptiles (the major source of endogenous auxins) attached. Auxin increased the duration of the lag phase before anthocyanin synthesis began and reduced the rate during the subsequent linear phase. Elongation continued longer with IAA than without it and anthocyanin formation did not begin until extension growth had ceased or was slowing down in both cases; the rate of anthocyanin synthesis in the IAA solution remained depressed compared with that in buffer even after extension growth had ceased in both.At low concentrations IAA stimulated elongation growth without reducing anthocyanin yield and it is unlikely that the effect of IAA on anthocyanin synthesis results from the increased utilisation in growth of substrates needed for anthocyanin formation. The results of reciprocal transfer experiments from dark to light, and vice versa, showed that the action of IAA was associated with its presence in the incubating medium during the irradiation period. If present only in darkness, before or after transfer to light, IAA did not reduce anthocyanin formation; in the former case total yield was increased by IAA as a result of the stimulation of elongation growth, the concentration of anthocyanin remaining unchanged.GA₃ also decreased anthocyanin content; the effect was greater in sections incubated with coleoptiles attached and it is possible that GA₃ acts by increasing the concentration of endogenous auxins. However, CCC, which has been reported to decrease endogenous auxin levels, also reduced anthocyanin yield.The effect of IAA was not influenced by the presence of ascorbate in the incubating medium, nor did added ascorbate result in the formation of any acylated cyanidin derivative in internodes maintained in darkness.Possible relationships between light-induced anthocyanin formation and the photo-inhibition of elongation are discussed.     

The Relationship between Growth and Proline Incorporation after Auxin Treatment of Mung Bean Hypocotyls

Indoleacetic acid (IAA) stimulates the incorporation of ¹⁴C-proline into both the cyloplasmic and the cell wall fractions of the hypocotyl of mung bean (Phaseolus aureus Roxb. cv. Black). It neither stimulates the transfer of ¹⁴C-proline from the cyloplasmic fraction into the cell wall fraction, nor the retention of ¹⁴C-proline in the wall or cytoplasmic fractions. Moreover, the stimulation of growth caused by IAA parallels the stimulation of the incorporation of proline into the cytoplasmic fraction, but does not parallel the stimulation into the cell wall fractions. The stimulation of the incorporation into the cyloplasmic fraction seems to appear within 30 minutes after auxin treatment, at about the same time the increase in the growth is observed in response to IAA, suggesting a connection between these effects. On the other hand, the stimulation of the proline incorporation into the cell wall fraction seems to require more than 90 minutes after auxin treatment, suggesting no close connection between growth and proline incorporation into the cell wall fraction.     

Changes in auxin patterns in developing gynophores of the peanut plant (Arachis hypogaea L.)

The peanut plant (Arachis hypogaea L.) produces flowers aerially, but buries the recently fertilized ovules in the soil in order for the fruit and seeds to mature underground. The organ that carries the seeds into the soil is called the gynophore. The growth of the peanut gynophore is regulated primarily by indole-3-acetic acid (IAA). A monoclonal antibody raised against IAA was used to successfully detect and localize this growth substance in the tissues of developing peanut gynophores. Five different stages of development were analysed: (1) before fertilization; (2) after fertilization; (3) during downwards growth; (4) at soil penetration; and (5) at the early stages of fruit formation. While no auxin signal is visible in the unfertilized ovules and ovary region, an asymmetric signal is observed in the gynophore wall after fertilization. During downwards growth, the auxin signal is located in both the meristematic region and in the area encircling the seeds, as well as in the cortex and epidermis region of the elongation zone. Upon soil penetration, the auxin signal in the meristematic region disappears, and most of the signal is detected in the gynophore wall near the tip. At the early stages of peanut fruit development, auxin signal is found at the lowermost area of the bending fruit, which eventually causes the fruit to be positioned horizontally. The results of this study suggest that the possible source of auxin within the gynophore may be the area of the gynophore wall close to the tip.     

Protein synthesis and wall extensibility in the Avena coleoptile

Summary The inhibitors cycloheximide and puromycin have been used to examine the relationship between protein synthesis and wall extensibility, as measured with an Instron, in Avena coleoptile segments. Cycloheximide at 4 g/ml almost totally inhibits both auxin-induced cell elongation and protein synthesis with only a slight lag. Wall extensibility is unaffected by the inhibitor if auxin is absent. If added prior to auxin, cycloheximide prevents auxin-induced wall loosening while if added after auxin it causes a substantial decline in the wall extensibility. With puromycin there is a 2–4 hr lag before growth and wall loosening are inhibited. These results support the conclusions that the proteins needed for wall loosening are unstable, and that continued protein synthesis is necessary to maintain the wall loosening process.     

Ethylene and auxin-induced cell growth in relation to auxin transport and metabolism and ethylene production in the semi-aquatic plant,Regnellidium diphyllum

Cell elongation in the rachis of the semiaquatic fern Regnellidium diphyllum is induced by the addition of ethylene or indoleacetic acid (IAA). Experiments with whole plants or rachis segments have shown that ethylene-induced growth requires the presence of auxin. Ethylene does not cause a modification in either endogenous auxin levels or in the extent of auxin metabolism but auxin transport is reduced. Rates of ethylene production in Regnellidium are not altered by either mechanical excitation or by the addition of auxin. A two-hormone control of cell expansion is proposed in which an initial, auxin-dependent growth event pre-conditions the cells to a further subsequent (or synchronous) ethylene-dependent growth event.Abbreviation IAA indole-3yl-acetic acid     

Transport of Indole-3-Acetic Acid during Gravitropism in Intact Maize Coleoptiles

We have investigated the transport of tritiated indole-3-acetic acid (IAA) in intact, red light-grown maize (Zea mays) coleoptiles during gravitropic induction and the subsequent development of curvature. This auxin is transported down the length of gravistimulated coleoptiles at a rate comparable to that in normal, upright plants. Transport is initially symmetrical across the coleoptile, but between 30 and 40 minutes after plants are turned horizontal a lateral redistribution of the IAA already present in the transport stream occurs. By 60 minutes after the beginning of the gravitropic stimulus, the ratio of tritiated tracer auxin in the lower half with respect to the upper half is approximately 2:1. The redistribution of growth that causes gravitropic curvature follows the IAA redistribution by 5 or 10 minutes at the minimum in most regions of the coleoptile. Immobilization of tracer auxin from the transport stream during gravitropism was not detectable in the most apical 10 millimeters. Previous reports have shown that in intact, red light-grown maize coleoptiles, endogenous auxin is limiting for growth, the tissue is linearly responsive to linearly increasing concentrations of small amounts of added auxin, and the lag time for the stimulation of straight growth by added IAA is approximately 8 or 9 minutes (TI Baskin, M Iino, PB Green, WR Briggs [1985] Plant Cell Environ 8: 595-603; TI Baskin, WR Briggs, M Iino [1986] Plant Physiol 81: 306-309). We conclude that redistribution of IAA in the transport stream occurs in maize coleoptiles during gravitropism, and is sufficient in degree and timing to be the immediate cause of gravitropic curvature.     

Evidence that Auxin-induced Growth of Soybean Hypocotyls Involves Proton Excretion

The role of H⁺ excretion in auxin-induced growth of soybean hypocotyl tissues has been investigated, using tissues whose cuticle was rendered permeable to protons or buffers by scarification (scrubbing). Indoleacetic acid induces both elongation and H⁺ excretion after a lag of 10 to 12 minutes. Cycloheximide inhibits growth and causes the tissues to remove protons from the medium. Neutral buffers (pH 7.0) inhibit auxin-induced growth of scrubbed but not intact sections; the inhibition increases as the buffer strength is increased. Both live and frozen-thawed sections, in the absence of auxin, extend in response to exogenously supplied protons. Fusicoccin induces both elongation and H⁺ excretion at rates greater than does auxin. These results indicate that H⁺ excretion is involved in the initiation of auxin-induced elongation in soybean hypocotyl tissue.     

Growth Inhibition and Recovery in Roots Following Temporary Treatment with Auxin

Continuous recording (streak photography) of elongation of roots treated with IAA (10^–6–10^–7M) showed that removal of IAA from the nutrient solution resulted in a rapid resumption of elongation, unless the IAA treatment was shorter than 60 min. If it was shorter, the recovery was delayed, so that it occurred about 1 hour after the beginning of the treatment, independently of the duration of the treatment, down to 4 min. This behavior of roots was observed in all the species investigated (corn, pea, sunflower, onion), also in response to NAA and 2,4-D. This time lag in recovery of elongation after brief auxin treatment is discussed in connection with the radial concentration gradient of auxin in the root imposed by external auxin. The possible role of a radial gradient of auxin (concentration decreasing with distance from the center) in the control of root elongation is suggested.     

Auxin Asymmetry during Gravitropism by Tomato Hypocotyls

Gravitropic asymmetry of auxin was observed in hypocotyls of tomato (Lycopersicon esculentum Mill.) soon after horizontal placement: the ratio of apically supplied [³H]IAA collected from the lower sides to that from the upper sides was about 1.4 between 5 and 10 minutes. This was adequately early to account for the beginning of curvature. The auxin asymmetry ratio rose to about 2.5 between 20 and 25 minutes, and to 3.5 during the main phase of curvature. This compares reasonably well with the roughly 3.9 ratio for elongation on the lower side to elongation on the upper side that is the basis for the curvature. These data extend evidence that the Went-Cholodny theory for the mediation of tropisms is valid for dicot stems. Also consistent with the theory, an auxin asymmetry ratio of 2.5 was observed when wrong-way gravitropic curvature developed following application of a high level of auxin. In addition to reversing the asymmetry of elongation, the large supplement of auxin resulted in lower net elongation. Previous data established that ethylene is not involved in this decrease of growth as a function of increasing level of auxin.     

The effect of cytochalasin B on the rate of growth and ultrastructure of wheat coleoptiles and maize roots

Cytochalasin B (CB) inhibits the elongation growth of maize roots, and that of wheat coleoptile segments incubated in indolyl-3-acetic acid, by over 30% after a lag period of about 60 min. This long lag is not due to poor tissue penetration by the inhibitor, but seems to reflect a property of the process inhibited by CB. The only visible ultrastructural change accompanying growth inhibition is the accumulation of secretory vesicles in the vicinity of dictyosomes, which occurs between 90 and 300 min. However, a massive accumulation of vesicles is seen after 120 min in root cap cells which possess very active dictyosomes. The results indicate that CB does not inhibit elongation growth by interfering with cytoplasmic streaming. Instead, they indicate that the drug acts to inhibit the secretion of cell wall components at some stage after vesicle production, but prior to their transport.Abbreviations CB cytochalasin B - IAA indolyl-3-acetic acid - DMSO dimethyl sulphoxide     

The Effect of Coumarin on Young's Modulus in Sunflower Stems and Maize Roots and on Water Permeability in Potato Parenchyma

By means of the resonance frequency method Young's, modulus has been determined after coumarin treatment of growing segments of etiolated sunflower hypocotyl segments and in maize roots. Coumarin caused a decrease in Young's modulus in both shoot and root tissue. The response was very rapid; in sunflower hypocotyls the decrease in elastic modulus appeared 3 min after application of coumarin. The effects produced by coumarin were similar to those found by auxin. Coumarin increased the rate of water efflux out of potato parenchyma by about 20%. The increase in water permeability was evident within 3 min.