A New Sensitive Root Auxanometer: Preliminary Studies of the Interaction of Auxin and Acid pH in the Regulation of Intact Root Elongation

A new sensitive root auxanometer is described. The auxanometer represents an adaption of the position-sensor transducer method to measurement of intact root elongation and has the advantages of simplicity and high sensitivity. Experiments with the auxanometer show that auxin begins to inhibit intact pea root elongation within 10 minutes and continues to inhibit elongation for at least 1 hour following a 1-hour treatment with the hormone. Exposure of pea roots to pH 4 results in a 2- to 3-fold increase in elongation rate beginning about 1 minute after acid treatment. Acid-induced elongation continues at a steady rate for at least 160 minutes and can be reinitiated repeatedly by shifting between pH 4 and 6.5. Auxin inhibits acid-induced elongation whether given before or after acidification, and a transient exposure to auxin renders intact roots relatively insensitive to acid for at least 1 hour after withdrawal of the hormone.     

Polar auxin transport and auxin-induced elongation in the absence of cytoplasmic streaming

Summary When cytoplasmie streaming in oat and maize coleoptile cells is completely inhibited by cytochalasin B (CB), polar transport of auxin (indole-3-acetic acid) continues at a slightly reduced rate. Therefore, cytoplasmic streaming is not required for polar transport. Auxin induces elongation in CB-inhibited coleoptile and pea stem segments, but elongation rate is reduced about 40% by CB. Therefore, stimulation of cytoplasmic streaming cannot be the means by which auxin promotes cell elongation, but streaming may be beneficial to elongation growth although not essential to it. A more severe inhibition of elongation develops after several hours in CB. With coleoptiles this could be due to inhibition of sugar uptake; in pea tissue it may be due to permeability changes and cytoplasmic degeneration. CB does not disorganize or disorient microfilament bundles when it inhibits streaming in maize, but appears instead to cause hypercondensation of microfilament material.     

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.     

Mechanism of Auxin-induced Ethylene Production

Indoleacetic acid-induced ethylene production and growth in excised segments of etiolated pea shoots (Pisum sativum L. var. Alaska) parallels the free indoleacetic acid level in the tissue which in turn depends upon the rate of indoleacetic acid conjugation and decarboxylation. Both ethylene synthesis and growth require the presence of more than a threshold level of free endogenous indoleacetic acid, but in etiolated tissue the rate of ethylene production saturates at a high concentration and the rate of growth at a lower concentration of indoleacetic acid. Auxin stimulation of ethylene synthesis is not mediated by induction of peroxidase; to the contrary, the products of the auxin action which induce growth and ethylene synthesis are highly labile.     

Comparison of Auxin-induced and Acid-induced Elongation in Soybean Hypocotyl

Acid-induced growth was compared to auxin-induced growth. After a transient pH 4-induced increase in the elongation rate was completed, auxin could still induce an enhanced rate of elongation in soybean (Glycine max) hypocotyl segments. This auxin response occurred both when the medium was changed to pH 6 before auxin addition, and when the auxin was added directly to the pH 4 medium. This postacid response to auxin was persistent, and quite unlike a postacid response to acid, which was again shortlived. One mm N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7) inhibited the first response to auxin (the first response to auxin being similar to the acid response), but not the second response. This did not appear to be simply a hydrogen ion neutralizing effect, however, since a 50-fold increase in buffer concentration at pH 6 did not inhibit the first response. Decrease in the pH of the external medium, previously shown to occur with excised soybean hypocotyl segments, was not affected by auxin. Furthermore, this pH drop, during which the cells appear to be adjusting their external pH to about 5.4, did not result in an increased rate of elongation. Addition of auxin after the equilibrium pH had been attained did not alter the pH, but it did increase the rate of elongation, eliciting a normal auxin response. It was concluded that hydrogen ions do not mediate in long term auxin-induced elongation in soybean hypocotyl.     

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.     

Auxin-induced elongation of short maize coleoptile segments is supported by 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one

Endogenous extractable factors associated with auxin action in plant tissues were investigated, especially their effects on elongation of 1-mm coleoptile segments of maize (Zea mays L.), in the presence of saturating 10 μM indole-3-acetic acid (IAA). The relative growth response, to auxin alone, was much smaller in segments shorter than 2–3 mm compared to 10-mm segments. Fusicoccin-induced elongation, however, was less affected by shortening the segments. A reduced auxin response may result from the depletion through cut surfaces of a substance required for IAA-mediated growth. Sucrose, phenolics like flavonoids, and vitamins were ruled out as the causal factors. A partially purified methanol extract of maize coleoptiles supported long-term, auxin-controlled elongation. The active material was also found among substances bleeding from scrubbed maize coleoptiles. The active factor from maize was further purified by HPLC and characterised by the UV spectrum and its pH shift. This factor was identified as 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) by mass spectroscopy. Activity tests confirmed that pure DIMBOA from other sources sustained auxin-induced elongation of short maize coleoptile segments. However, DIMBOA only partially restored the activity lost from short segments. This indicates that an additional factor, other than DIMBOA, is required. Extracts from Avena or Cucurbita did not contain the factor DIMBOA; it was active on maize elongation, but not on Avena coleoptiles or Cucurbita hypocotyls. This narrow specificity and the lack of DIMBOA action in short-term tests with maize indicate that DIMBOA is not the general auxin cofactor but may specifically “spare” the co-auxin in maize. Received: 27 June 2000 / Accepted: 16 October 2000     

Timing of the auxin response in etiolated pea stem sections

The short term growth response of etiolated pea stem segments (Pisum sativum L., var. Alaska) was investigated with the use of a high resolution growth-recording device. The immediate effect of treatment with indole-3-acetic acid is an inhibition of growth. This inhibition lasts about 10 minutes, and then the rate of elongation rises abruptly to a new steady rate about 4 times the rate of elongation before auxin treatment. This rapid steady rate of elongation, however, continues for only about 25 minutes before declining suddenly to a lower steady rate of growth about 2 times the rate of elongation before the addition of auxin. Pretreatment of the segments with cycloheximide or actinomycin strongly inhibits both phases of auxin-promoted elongation without altering the length of the latent period in response to the hormone.     

Selective effects of victorin on growth and the auxin response in Avena

Victorin, the pathotoxin from the host-specific pathogen, Helminthosporium victoriae, promotes the growth of coleoptile segments when given at concentrations that are high but which still show selective effects on susceptible and resistant tissue. The latent period in the growth response of both susceptible and resistant tissue is about 3.6 minutes compared to 11.0 minutes in the response of these tissues to auxin. The victorinpromoted rate of elongation of 8-millimeter segments is about 0.2 millimeter per hour in susceptible tissue and about 0.1 millimeter per hour in resistant tissue compared to about 0.4 millimeter per hour in response to auxin. At low concentrations, the toxin has no growth-promoting effect in either susceptible or resistant coleoptile segments. Over a wide range of concentrations, victorin inhibits the growth response of susceptible tissue to auxin completely while having no effect on the response of resistant tissue to auxin.     

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.     

Auxin-induced H Secretion in Helianthus and Its Implications

We have examined the ability of Helianthus hypocotyl segments as well as segments from a variety of other species to elongate in response to H⁺ and to secrete H⁺ in response to auxin and fusicoccin. In all cases a positive response was obtained when the cuticular barrier was abraded with carborundum. Removal of the cuticular barrier by “peeling” prevented detection of both auxin-induced elongation and H⁺ secretion. Fusicoccin-induced growth and acid secretion are not prevented by peeling. These results suggest considerable tissue selectivity with respect to auxin action but considerably less specificity with respect to fusicoccin. It seems likely that in many dicots auxin-enhanced proton secretion and elongation are controlled by the epidermis and/or closely associated cell layers. The data presented in this paper provide further support for the acid growth theory of auxin action.     

Control of Auxin-induced Stem Elongathn by the Epidermis

Segments of the 4th and 5th internodes of light-grown pea seedlings were used for the study of control of stem elongation. With 5th internodes, at low turgor as well as at water saturation auxin primarily appeared to cause a change in cell wall properties of the epidermis but it showed little effect on expansion af the inner tissue. This was confirmed by comparison of expansion between peeled and unpeeled segments, split tests and by measurements of stress-relaxation properties of the epidermal cell wall. Segments with the central part re-moved elongated well in response to auxin, but the isolated epidermis showed neither auxin-induced elongation nor cell wall loosening. A fungal β-1,3-glucanase appeared, at least partly, to have a similar effect as that of auxin on elongation, by changing cell wall properties of the epidermal cell wall. Peeled segments of 4th internodes expanded very little and auxin had little effect on their epidermal cell wall properties.     

The pH profile for acid-induced elongation of coleoptile and epicotyl sections is consistent with the acid-growth theory

The acid-growth theory predicts that a solution with a pH identical to that of the apoplast of auxintreated tissues (4.5–5.0) should induce elongation at a rate comparable to that of auxin. Different pH profiles for elongation have been obtained, however, depending on the type of pretreatment between harvest of the sections and the start of the pH-incubations. To determine the acid sensitivity under in vivo conditions, oat (Avena sativa L.) coleoptile, maize (Zea mays L.) coleoptile and pea (Pisum sativum L.) epicotyl sections were abraded so that exogenous buffers could penetrate the free space, and placed in buffered solutions of pH 3.5–6.5 without any preincubation. The extension, without auxin, was measured over the first 3 h. Experiments conducted in three laboratories produced similar results. For all three species, sections placed in buffer without pretreatment elongated at least threefold faster at pH 5.0 than at 6.0 or 6.5, and the rate elongation at pH 5.0 was comparable to that induced by auxin. Pretreatment of abraded sections with pH-6.5 buffer or distilled water adjusted to pH 6.5 or above gave similar results. We conclude that the pH present in the apoplast of auxin-treated coleoptile and stems is sufficiently low to account for the initial growth response to auxin.Abbreviations FS free space - IAA indole-3-acetic acid This research was supported by a grant from the National Adonautics and space Administration (NASA), NAGW 1394 to R.E.C., NASA grant NAGW-297 to M.L.E., and NASA grant NAG 1849 to D.L.R.     

Growth and development, and auxin polar transport in higher plants under microgravity conditions in space: BRIC-AUX on STS-95 space experiment.

The principal objectives of the space experiment, BRIC-AUX on STS 95, were the integrated analysis of the growth and development of etiolated pea and maize seedlings in space and a study of the effects of microgravity conditions in space on auxin polar transport in these segments. Microgravity significantly affected the growth and development of etiolated pea and maize seedlings. Epicotyls of etiolated pea seedlings were the most oriented toward about 40 to 60 degrees from the vertical. Mesocotyls of etiolated maize seedlings were curved at random during space flight but coleoptiles were almost straight. Finally the growth inhibition of these seedlings in space was also observed. Roots of some pea seedlings grew toward to the aerial space of Plant Growth Chamber. Extensibilities of cell walls of the third internode of etiolated pea epicotyls and the top region of etiolated maize coleoptiles, which were germinated and grown under microgravity conditions in space, were significantly low as compared with those grown on the ground of the earth. Activities of auxin polar transport in the second internode segments of etiolated pea seedlings and coleoptile segments of etiolated maize seedlings were significantly inhibited and promoted, respectively, under microgravity conditions in space. These results strongly suggest that auxin polar transport as well as the growth and development of plants is controlled under gravity on the earth.     

Control and Kinetics of Branch Root Formation in Cultured Root Segments of Haplopappus ravenii

Branch root formation required only the presence of minerals, sucrose as a carbon source, and an auxin. The number of primordia formed was a function of auxin concentration. With naphthaleneacetic acid at 0.1 mg/l, up to 60 or more branches were formed per centimeter of Haplopappus ravenii root segment. Under our conditions, pea root segments formed only five or six branches per centimeter, but tomato and radish, like H. ravenii, formed large numbers of branches. Cytokinin inhibited branch formation, while gibberellic acid was without effect. Vitamins were not required for branch formation, although they enhanced elongation. Up to 5 days were required for the maximum number of stable branch primordia to form under the influence of naphthaleneacetic acid. If naphthaleneacetic acid was withdrawn earlier, fewer branch primordia developed. The requirement for a lengthy exposure to naphthaleneacetic acid, the kinetics of the response, and the ease with which naphthaleneacetic acid could be rinsed out of the tissue with consequent cessation of branch root formation, were similar to other hormone-regulated developmental systems. Anatomical and cytological studies were made of segments exposed for various times to auxin. The segments were mostly diarch, and branches formed obliquely to protoxylem poles. While primarily only pericycle-endodermis cells divided, both these and cortex cells responded in the first 24 hours exposure to naphthaleneacetic acid with enlarged nuclei and nucleoli, and a few cortical cells divided. Maximum nucleus and nucleolus size was reached approximately 9 hours after exposure to naphthaleneacetic acid. Branches rarely elongated more than 5 cm before their meristems died. The H. ravenii culture is maintained only by the frequent formation of new naphthaleneacetic acid-induced branches.     

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.     

Rapid Production of Auxin-induced Ethylene

The time course of auxin-induced ethylene production was determined in mesocotyl segments of etiolated sorghum (Sorghum bicolor L. Moench) seedlings. The latent period between addition of auxin and a detectable rise in ethylene release was 15 to 20 minutes in four different genotypes. This may indicate that the initial effect of auxin on ethylene production is too rapid to involve synthesis of an ethylene-producing enzyme. The technique devised for these experiments involves placing tissue segments end to end in a glass tube, and it allows simultaneous determination of growth and ethylene production.     

Evidence for Receptor Function of Auxin Binding Sites in Maize : RED LIGHT INHIBITION OF MESOCOTYL ELONGATION AND AUXIN BINDING

When 3- to 4-day-old dark-grown maize (Zea mays L. WF9 × Bear 38) seedlings are given red light, auxin-binding activity localized on endoplasmic reticulum membranes of the mesocotyl begins to decrease after 4 hours; by 9 hours, it falls to 50 to 60% of that in dark controls, on either a fresh weight or total particulate protein basis. Endoplasmic reticulum-localized NADH:cytochrome c reductase activity decreases in parallel. Loss of binding is due to decrease in number of sites, with no change in their affinity for auxin (K_d 0.2 micromolar for naphthalene-1-acetic acid). Elongation of mesocotyl segments in response to auxin decreases with a similar time course. Elongation of segments from irradiated plants shows the same apparent affinity for auxin as that of the dark controls. Auxin-binding activity and elongation response also decrease in parallel down the length of the mesocotyl. These observations are consistent with a role of endoplasmic reticulum-localized auxin binding sites as receptors for auxin action in cell elongation.     

STS-95 space experiment for plant growth and development, and auxin polar transport.

The principal objective of the space experiment, BRIC-AUX on STS-95, was the integrated analysis of the growth and development of etiolated pea and maize seedlings in space, and the effect of microgravity conditions in space on auxin polar transport in the segments. Microgravity conditions in space strongly affected the growth and development of etiolated pea and maize seedlings. Etiolated pea and maize seedlings were leaned and curved during space flight, respectively. Finally the growth inhibition of these seedlings was also observed. Roots of some pea seedlings grew toward the aerial space of Plant Growth Chamber. Extensibilities of cell walls of the third internode of etiolated pea epicotyls and the top region of etiolated maize coleoptiles which were germinated and grown under microgravity conditions in space were significantly low. Activities of auxin polar transport in the second internode segments of etiolated pea seedlings and coleoptile segments of etiolated maize seedlings were significantly inhibited and extremely promoted, respectively, under microgravity conditions in space. These results strongly suggest that auxin polar transport as well as the growth and development of plants is controlled under gravity on the earth.     

Effects of Exogenously Applied Jasmonates on Growth and Intracellular pH in Maize Coleoptile Segments

The effects of jasmonic acid (JA) on elongation growth of coleoptile segments from etiolated maize (Zea mays L.) were investigated in the presence and absence of auxin. When supplied alone, at physiological concentrations (10⁻⁹, 10⁻⁸, and 10⁻⁵ m), JA (or methyl-JA) inhibited growth. JA at a similar range of concentrations also inhibited auxin-induced elongation growth. To determine whether this effect on growth depended on endogenous abscisic acid (ABA), we grew maize coleoptiles in the presence of norflurazon (an inhibitor of carotenoid biosynthesis) that results in reduced endogenous ABA levels. Growth of etiolated coleoptile segments from these plants was inhibited by JA (or methyl-JA) in both the absence and presence of auxin. Previously, we have observed a correlation between elongation growth and cytosolic pH (pH_i), in which auxin lowers pH_i, and growth inhibitors such as ABA raise pH_i. We examined the effect of low concentrations of methyl-JA on pH_i with dual emission dye, carboxy seminaphthorhodafluor-1, and confocal microscopy. To confirm these studies, we also used in vivo ³¹P NMR spectrometry to ascertain the changes in pH_i after addition of jasmonate to maize coleoptiles. Coleoptiles grown in either the absence or presence of norflurazon responded to methyl-JA or JA by increases in pH_i of approximately 0.2 pH unit. This response occurs over a period of 15–20 min and appears to be independent of endogenous ABA. This alkalization induced by JA is likely to form a permissive environment for JA signal transduction pathway(s). Received February 5, 1999; accepted August 25, 1999