Differential effect of auxin on in vivo extensibility of cortical cylinder and epidermis in pea internodes

The effect of auxin indole-3-acetic acid (IAA) on growth and in vivo extensibility of third internode sections from red light grown pea seedlings (Pisum sativum L. cv Alaska) and the isolated tissues (cortex plus vascular tissue = cortical cylinder, and epidermis) was investigated. Living tissue was stretched at constant force (creep test) in a custom-built extensiometer. In the intact section, IAA-induced increase in total (E_tot), elastic (E_el), and plastic (E_pl) extensibility is closely related to the growth rate. The extensibility of the cortical cylinder, measured immediately after peeling of intact sections incubated for 4 hours in IAA, is not increased by IAA. Epidermal strips, peeled from growing sections incubated in IAA, show a E_pl increase, which is correlated to the growth rate of the intact segments. The isolated cortical cylinder expands in water; IAA has only a small growth-promoting effect. The extensibility of the cortical cylinder is not increased by IAA. Epidermal strips contract about 10% on isolation. When incubated in IAA, they do not elongate, but respond with an E_pl increase. The amount of expansion of the cortical cylinder and contraction of the epidermis (tissue tension), measured immediately following excision and peeling, stays constant during IAA-induced growth of intact sections. The results support the hypothesis that IAA induces growth of the intact section by causing an E_pl increase of the outer epidermal wall. The driving force comes from the expansion of the cortical cylinder which is under constant compression in the intact section.     

Auxin concentration/growth relationship for Avena coleoptile sections from seedlings grown in complete darkness

A biphasic auxin dose-response curve has been obtained for indole-acetic acid (IAA)-stimulated growth of subapical sections of coleoptiles from totally dark-grown oats (Avena sativa L. cv Lodi). The curve for growth at 6 h is composed of a log-linear phase and a modified bell-shaped phase separated by a plateau. The curve is log-linear from 0.003 to 0.4 micromolar IAA when sections are incubated in pH 5.9 buffer. The plateau of IAA concentration-neutral growth is seen from 0.4 to 4.0 micromolar IAA. Further increase in growth occurs from 4.0 to 10 micromolar IAA. Changing the pH of the buffer from 5.9 to 5.5 or 6.2 changes the shape of the curve, shifting the plateau to lower IAA concentration, or abolishing it, respectively. The synthetic auxin 2,4-dichlorophenoxyacetic acid also shows a biphasic dose-response curve, but the synthetic auxin 1-naphthalene acetic acid does not. The plateau is not affected by the auxin-transport inhibitor 2,3,5-triiodobenzoic acid. The plateau is eliminated by taking sections from coleoptiles grown under continuous dim red light. We advance a model to account for these results based on two modes of auxin uptake into the cell: carrier-mediated uptake and uptake via chemiosmotic diffusion.     

Blockage by gibberellic Acid of phytochrome effects on growth, auxin responses, and flavonoid synthesis in etiolated pea internodes

Red light inhibits the growth of etiolated pea internodes, causes a shift toward higher indoleacetic acid (IAA) concentrations in the IAA dose-response curve of excised sections, and promotes the synthesis in intact internodes of kaempferol-3-triglucoside. Gibberellic acid (GA₃) prevents all 3 effects, the first effect substantially and the last 2 completely. This suggests GA₃ blockage of an early or basic event initiated by the active form of phytochrome. The red light-induced shift in the IAA dose-response curve of excised sections is consistent with a light-induced increase in the activity of an IAA destruction system, since the magnitude of the red light inhibition varied with IAA concentration. The red light and GA₃ effects on growth and on flavonoid synthesis are consistent with the view that phytochrome may control growth by regulating the synthesis of phenolic compounds which act as cofactors in an IAA-oxidase system. GA₃ reversal of the red light-induced shift in the IAA dose-response curve involves both growth promotion and inhibition by GA₃ at different IAA concentrations and this, together with the GA₃ reversal of light-induced flavonoid synthesis, supports the suggested regulatory role of phenolic compounds in growth.     

Short-term kinetics of elongation growth of gibberellin-responsive lettuce hypocotyl sections

The short-term kinetics of growth of the excised lettuce (Lactuca sativa L.) hypocotyl were characterized with respect to the effects of gibberellic acid (GA₃), indole-3-acetic acid (IAA), KCl and pH. A Hall-device-based, miniaturized, linear displacement transducer was developed to measure the growth of 2-mm hypocotyl sections with 1-m resolution. Following treatment with GA₃, a lag time of less than 10 min was typically followed by an increase in growth rate with two acceleration phases, reaching a final elevated rate within about 1 h. The kinetics of the response to GA₁, a mixture of GA₄ and GA₇, and GA₉ were similar to the response to GA₃. There was no response to IAA treatment either in the presence or absence of GA₃. KCl alone had no effect on the growth rate, but caused an increase in rate when added after GA₃, with a lag time of usually less than 1 h. Responses to pH changes had lag times of a few minutes in all cases. A shift from H₂O to pH 6 buffer inhibited growth, while a shift from H₂O to pH 4 buffer resulted in a transient increase to a rate comparable to that induced by GA₃. A shift from pH 6 to pH 5 caused an increase in growth rate, followed by a gradual decline to an H₂O control rate after more than an hour. The responses to GA₃ at pH 4 and pH 5 were similar to that found for addition of GA₃ to water controls.Abbreviations GA gibberellin - GA₃ gibberellic acid - GA₁, GA₄₊₇, GA₉ gibberellins A₁, A₄₊₇, A₉ - IAA indole-3-acetic acid     

Interaction between cortical cylinder and epidermis during auxin-mediated growth in pea internodes

The interaction between cortical cylinder (cortex plus vascular tissue) and epidermis during auxin (indole-3-acetic acid, IAA)-induced growth of third internode sections from red light-grown pea seedlings (Pisum sativum L. cv. Alaska) was investigated. A quantitative comparison of the relative effects of IAA on growth of intact and peeled sections showed that intact segments are nearly 20-fold more sensitive to IAA than peeled cortical cylinders. Tissue tension, determined with the ‘split section test’, was constant during IAA-induced growth of intact sections. Peeled sections also displayed a small amount of tissue tension, which was likewise independent of IAA. The incorporation of myo-[2-³H(N)]inositol ([³H]Ins) into non-cellulosic polysaccharides in the cell walls was stimulated by IAA in both the cortical cylinder and the epidermis by + 70% and + 55%, respectively, after 4 h. A mich higher amount of incorporation was detectable in the epidermis than in the cortical cylinder on a unit weight basis. During a 4-h growth period in IAA the cortical cylinder lost about 50 μg of its initial dry weight per section whereas the epidermis increased in dry weight by about + 24 μg. We conclude that during IAA-induced long-term growth the cortical cylinder (1) provides the driving force for organ growth, (2) responds to IAA by an increase in matrix cell wall synthesis and (3) releases material, some of which is transferred to the attached epidermal cells.     

Uptake and decarboxylation of indole-3-acetic acid during auxin-induced growth in lupin hypocotyl segments

The elongation growth of etiolated hypocotyl segments of lupin (Lupinus albus L.) was stimulated by acid pH (4.6 versus 6.5) and by IAA for periods of up to 4 h. After this time, the segments were unable to grow further. In the presence of an optimal IAA concentration (10 μM), acid pH increased the growth rate but had no effect on final growth. With suboptimal IAA (0.1 μM), however, acid pH increased growth in a more than additive way, suggesting a synergistic action between the two factors. This synergism may be explained by the increased IAA uptake and decarboxylation seen at an acid pH. These results reinforce the view that the effects of low pH and IAA on growth are not independent. Vanadate inhibited growth and also IAA uptake and decarboxylation. This inhibitor, therefore, probably inhibits growth not only by decreasing ATPase-mediated acidification but also by decreasing H⁺-dependent IAA uptake from the apoplasm. This dependence of IAA uptake on ATPase may be mediated by apoplasmic acidification. The amount of IAA decarboxylated increased when the assay conditions favored the growth of segments, indicating that IAA could be destroyed by decarboxylation during the auxin-induced growth.     

Indoleacetic Acid-Induced Decrease of the Ribomiclease Activity in vivo

A study has been made on the influence of indole-3-acetic acid (IAA) on the ribonuclease (RNase) activity in wheat coleoptile sections and green pea stem sections. The hormonal effects on the enzyme activity, ribonncleic acid (RNA) metabolism and growth have been compared. Addition of 10^?5M IAA to the plant sections causes their RNase activity to decrease and their elongation to increase. Removal of the added IAA results in increasing enzyme activity and decreasing growth. The altered enzyme activities are paralleled by opposite changes in the RNA net synthesis. Administration of crystalline RNase to the plant tissue depresses growth. There is thus evidence that the in vivo effect of IAA on the RNase activity is of importance for the hormonal regulation of RNA metabolism and growth. The IAA-induced reduction in the enzyme activity involves cellular metabolism. The effect can be suspended by means of p-chloromercuribenzoate. A possible mechanism for the reduction is discussed.     

Effect of glyphosate on indole-3-acetic acid metabolism in tolerant and susceptible plants

A comparison study was conducted on the effect of glyphosate (N-[phosphonomethyl]glycine) on indole-3-[2-¹⁴C]acetic acid (IAA) metabolism, ethylene production, and growth of 7-day-old seedlings of different plants. The plants tested were American germander (Teucrium canadense L.), soybean (Glycine max L. Merr.), pea (Pisum sativum L. cv. Alaska and Little marvel), mungbean (Vigna radiata L.), and buckwheat (Fagopyrum esculentum Moench). A spray with 2 mM glyphosate affected IAA metabolism to a varied degree. The induced increase of IAA metabolism was greater in buckwheat, Alaska pea, and mungbean than soybean, Little marvel pea, and American germander. The increased IAA metabolism was correlated with the inhibition of growth and with the decrease of ethylene production. The natural rate of IAA metabolism was markedly different among the plant species and cultivars tested and appeared to be related to the sensitivity of the plants to glyphosate. American germander and Little marvel pea with high rates of IAA metabolism were more tolerant to glyphosate than buckwheat and Alaska pea, which had low rates of IAA metabolism. Plants with a high natural rate of IAA metabolism were probably less dependent on IAA and thus less susceptible to glyphosate.     

Release of lateral buds from apical dominance by glyphosate in soybean and pea seedlings

Application of a sublethal dose of glyphosate (N-[phosphonomethyl]glycine) to the seedlings of soybean (Glycine max L. Merr. cv. Evans) and pea (Pisum sativum L. cv. Alaska) promoted growth of the cotyledonary and other lateral buds. The pattern of the glyphosate-induced lateral bud growth was different from that induced by decapitation. Under the experimental condition, glyphosate did not kill the apical buds. Feeding stem sections of the seedlings with radiolabeled indole-3-acetic acid ([2¹⁴C]IAA) and subsequent analysis of free [2-¹⁴C]IAA and metabolite fractions revealed that the glyphosate-treated plants had higher rates of IAA metabolism than the control plants. The treated pea plants metabolized 75% of [2-¹⁴C]IAA taken up in the 4-h incubation period compared to 46.5% for the control, an increase of 61%. The increase was small but consistent in soybean seedlings. As a result, the glyphosate-treated plants had less free IAA and ethylene than the control plants. The increase of IAA metabolism induced by glyphosate is likely to change the auxin-cytokinin balance and contribute to the release of lateral buds from apical dominance in these plants.     

Auxin-Growth Relationships in Maize Coleoptiles and Pea Internodes and Control by Auxin of the Tissue Sensitivity to Auxin

Growth of a zone of maize (Zea mays L.) coleoptiles and pea (Pisum sativum L.) internodes was greatly suppressed when the organ was decapitated or ringed at an upper position with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) mixed with lanolin. The transport of apically applied ³H-labeled indole-3-acetic acid (IAA) was similarly inhibited by NPA. The growth suppressed by NPA or decapitation was restored by the IAA mixed with lanolin and applied directly to the zone, and the maximal capacity to respond to IAA did not change after NPA treatment, although it declined slightly after decapitation. The growth rate at IAA saturation was greater than the rate in intact, nontreated plants. It was concluded that growth is limited and controlled by auxin supplied from the apical region. In maize coleoptiles the sensitivity to IAA increased more than 3 times when the auxin level was reduced over a few hours with NPA treatment. This result, together with our previous result that the maximal capacity to respond to IAA declines in pea internodes when the IAA level is enhanced for a few hours, indicates that the IAA concentration-response relationship is subject to relatively slow adaptive regulation by IAA itself. The spontaneous growth recovery observed in decapitated maize coleoptiles was prevented by an NPA ring placed at an upper position of the stump, supporting the view that recovery is due to regenerated auxin-producing activity. The sensitivity increase also appeared to participate in an early recovery phase, causing a growth rate greater than in intact plants.     

Time-dependent Changes in the Auxin Sensitivity of Coleoptile Segments: Apparent Sensory Adaptation

When segments are excised from corn (Zea mays L.) coleoptiles they exhibit a very low rate of elongation for about 3.5 hours. A strong increase in growth rate (the spontaneous growth response) then occurs and persists for many hours. During the latent period preceding the spontaneous growth response there is an apparent increase with time in the sensitivity of the segments to indoleacetic acid (IAA). This increase in sensitivity is expressed as a 2- to 3-fold increase in the magnitude of the growth response to low levels of IAA and a 3-fold decrease in the latent period of the response during the first 3 hours following excision. A similar increase in sensitivity to low levels of IAA is noted if application of IAA is timed from the point of termination of a previous exposure to the hormone. Since the increase in responsiveness to low levels of IAA is not paralleled by an increase in the rate of uptake of the hormone, the data may be interpreted as evidence for a type of time-dependent sensory adaptation to auxin. The IAA dose-response relationship also changes with time, and there is indirect evidence that an auxin-dependent inhibitor may influence the expression of the apparent sensory adaptation to auxin.     

Nutrient salts promote light-induced degradation of indole-3-acetic Acid in tissue culture media

The disappearance of indole-3-acetic acid (IAA) from cell-free liquid culture medium was followed in response to nutrient salts found in Murashige-Skoog salt base, light, and pH range of 4 to 7. The loss of IAA was accelerated by light or Murashige-Skoog salts. However, the combination of both light and Murashige-Skoog salts acted synergistically to catalyze the destruction of over 80% of the original IAA within 7 days of continuous incubation. Under these same conditions, the loss of IAA was decreased to approximately 50% by adjusting the initial pH of the medium to 7. Iron was identified as the single major contributor to light-catalyzed destruction of IAA. Removal of nitrates, which represented 87% of the molar salt composition, also reduced the light-catalyzed loss of IAA. Treatments that protected IAA from degradation, such as darkness or removal of iron from the medium, suppressed the growth of muskmelon (Cucumis melo. Naud., var. reticulatus) callus tissue cultured for 30 days. Treatments in the light that rapidly degraded IAA resulted in maximum growth. Consequently, the brief exposure to IAA prior to degradation was apparently sufficient to initiate physiological changes required for growth. Possible approaches to the preservation of IAA during incubation are discussed.     

Comparison of the effect of indoleacetic Acid and fusicoccin on the breakdown of phosphatidylinositol in maize coleoptiles

The effect of indoleacetic acid (IAA) and fusicoccin (FC) on the breakdown of phosphatidylinositol in maize (Zea mays L.) coleoptiles has been studied. Coleoptiles were able to incorporate [³H] myo-inositol into the phospholipid fraction almost linearly for 8 hours. Thin layer chromatography analysis of total phospholipids showed that [³H]myo-inositol was incorporated only into phosphatidylinositol. Prelabeled coleoptiles treated with IAA showed a loss of the radioactivity incorporated in the phospholipid fraction, whose level decreased by 34% after 1 hour. Treatment with FC, on the contrary, did not modify the content of labelled phosphatidylinositol with respect to the control. The different effects of IAA and FC and a possible mechanism of IAA action on growth are discussed.     

Auxin Physiology of the Tomato Mutant diageotropica

The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxin-induced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.     

Effect of Deseeding on the Indole-3-acetic Acid Content of Shoots and Roots of Zea mays Seedlings

We wished to determine the effect of endosperm removal on the amounts of free and esterified indole-3-acetic acid (IAA) in young Zea mays seedlings. The increases of IAA derived from endosperm and from biosynthesis, but without correction for catabolic losses, were 0.9 picomole of free IAA per shoot per hour, and 1.1 picomoles per shoot per hour of ester IAA. After deseeding, free IAA in the shoot declines by 40% following kernel removal and total (free + ester) IAA declines at a rate of about 1 picomole per shoot per hour. A slight, but insignificant increase of ester IAA occurs following endosperm removal. In the primary roots, the decreases of free IAA and total (free + ester) IAA are accelerated by seed removal. Thus, the endosperm appears to be a major source of IAA for the shoot and root.     

Changes in Growth, Auxin- and Ribonucleic Acid Metabolism in Wheat Coleoptile Sections Following Pulse Treatment with Indole-3-Acetic Acid

Growth reactions of wbeat coleoptile sections following a brief pretreament in indole-3-acetic acid (LAA) were studied. The growth versus concentration curves 24 hours after the treatment showed a minimum value surrounded by bigber values. The minimum was never at concentrations lower than 10^-5M lAA but it could be found at higher concentrations after short pretreatment periods. The growth versus time curves reveated that the hormone treatment cansed the growth rate initially to increase but later on to decrease. The decrease was followed by a second increase for some treatments. Analysis of IAA content after the pretreatment showed that the attered growth patterns could be ascribed to declining auxin content with time, but not to thc actual concentration in the sections. The results indicate that the metabolic activation brought about by IAA leads to its own disappearance. Such a phenomenon was mirroretl in effects of IAA on hte net synthesis of ribonucleic acid.     

Inhibitory action of red light on the growth of the maize mesocotyl: evaluation of the auxin hypothesis

Brief irradiation of 3-d-old maize (Zea mays L.) seedlings with red light (R; 180 J m^-2) inhibits elongation of the mesocotyl (70–80% inhibition in 8 h) and reduces its indole-3-acetic acid (IAA) content. The reduction in IAA content, apparent within a few hours, is the result of a reduction in the supply of IAA from the coleoptile unit (which includes the shoot apex and primary leaves). The fluence-response relationship for the inhibition of mesocotyl growth by R and far-red light closely resemble those for the reduction of the IAA supply from the coleoptile. The relationship between the concentration of IAA (1–10 M) supplied to the cut surface of the mesocotyl of seedlings with their coleoptile removed and the growth increment of the mesocotyl, measured after 4 h, is linear. The hypothesis that R inhibits mesocotyl growth mainly by reducing the IAA supply from the coleoptile is supported. However, mesocotyl growth in seedlings from which the coleoptiles have been removed is also inhibited by R (about 25% inhibition in 8 h). This inhibition is not related to changes in the IAA level, and not relieved by applied IAA. In intact seedlings, this effect may also participate in the inhibition of mesocotyl growth by R. Inhibition of cell division by R, whose mechanism is not known, will also result in reduced mesocotyl elongation especially in the long term (e.g. 24 h).Abbreviations FR far-red light - IAA indole-3-acetic acid - P_fr phytochrome in the far-red-absorbing form - P_r phytochrome in the red-absorbing form - R red light     

Interaction of cell turgor and hormones on sucrose uptake in isolated Phloem of celery

Phloem tissue isolated from celery (Apium graveolens L.) was used to investigate the regulation of sucrose uptake by turgor (manipulated by 50-400 milliosomolal solutions of polyethylene glycol) and hormones indoleacetic acid (IAA) and gibberillic acid (GA₃). Sucrose uptake was enhanced under low cellular turgor (increase in the V_max). Furthermore, enhancement of sucrose uptake was due to a net increase in influx rates since sucrose efflux was not affected by cell turgor. Manipulations of cell turgor had no effect on 3-O-methyl glucose uptake. When 20 millimolar buffer was present in uptake solutions, low turgor-induced effects were observed only at low pH range (4.5-5.5). However, the effect was extended to higher external pH (up to 7.5) when buffer was omitted from uptake solutions. A novel interaction between cellular turgor and hormone treatments was observed, in that GA₃ (10 micromolar) and IAA (0.1-100 micromolar) enhanced sucrose uptake only at moderate turgor levels. The hormones elicited little or no response on sucrose uptake under conditions of low or high cell turgor. Low cell turgor, IAA, GA₃, and fusicoccin caused acidification by isolated phloem segments in a buffer-free solution. It is suggested that enhanced sucrose uptake in response to low turgor and/or hormones was mediated through the plasmalemma H⁺-ATPase and most likely occurred at the site of loading.     

The Growth-time Relationships of the Auxin- induced Growth in Avena Coleoptile Sections

1. 1. The growth rate of Avena coleoptile sections in the presenceof indoleacetic acid (IAA) is constant with time over a widerange of time intervals and IAA concentrations.
2. 2. Constancyof growth rate is dependent upon the maintenanceof constantconditions in which the concentration of IAA availableto thesection remains the chief factor limiting growth rate.
3. 3.Control of the pH of the medium in which the sections aregrownis essential to the maintenance of constant growth rate,particularlyin the presence of high concentrations of IAA.
4. 4. The lagperiod in establishment of steady growth rate bysections inthe presence of IAA is less than 10 minutes andis not detectableby present methods of measurement.

     

Auxin-induced leaf blade expansion in Arabidopsis requires both wounding and detachment

Elevation of leaf auxin (indole-3-acetic acid; IAA) levels in intact plants has been consistently found to inhibit leaf expansion whereas excised leaf strips grow faster when treated with IAA. Here we test two hypothetical explanations for this difference in growth sensitivity to IAA by expanding leaf tissues in vivo versus in vitro. We asked if, in Arabidopsis, IAA-induced growth of excised leaf strips results from the wounding required to excise tissue and/or results from detachment from the plant and thus loss of some shoot or root derived growth controlling factors. We tested the effect of a range of exogenous IAA concentrations on the growth of intact attached, wounded attached, detached intact, detached wounded as well as excised leaf strips. After 24 h, the growth of intact attached, wounded attached, and detached intact leaves was inhibited by IAA concentrations as little as 1 µM in some experiments. Growth of detached wounded leaves and leaf strips was induced by IAA concentrations as low as 10 µM. Stress, in the form of high light, increased the growth response to IAA by leaf strips and reduced growth inhibition response by intact detached leaves. Endogenous free IAA content of intact attached leaves and excised leaf strips was found not to change over the course of 24 h. Together these results indicate growth induction of Arabidopsis leaf blade tissue by IAA requires both substantial wounding as well as detachment from the plant and suggests in vivo that IAA induces parallel pathways leading to growth inhibition.