Effect of calmodulin antagonists on auxin-induced elongation

Coleoptile segments of oat (Avena sativa var Cayuse) and corn (Zea mays L. var Patriot) were incubated in different concentrations of calmodulin antagonists in the presence and absence of α-naphthaleneacetic acid. The calmodulin antgonists (chlorpromazine (CP), trifluoperazine, and fluphenazine) inhibited the auxin-induced elongation at 5 to 50 micromolar concentrations. Chlorpromazine sulfoxide, an analog of chlorpromazine, did not have significant effect on the elongation of oat and corn coleoptiles. A specific inhibitor of calmodulin N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide hydrochloride (W-7, a naphthalenesulfonamide derivative) inhibited coleoptile elongation, while its inactive analog N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) was ineffective at similar concentrations. During a 4-hour incubation period, coleoptile segments accumulated significant quantities of ³H-CP. About 85 to 90% of auxin-induced growth was recovered after 4 hours of preincubation with CP or 12 hours with W-7 and transferring coleoptiles to buffer containing NAA. Leakage of amino acids from coleoptiles increased with increasing concentration of CP, showing a rapid and significant increase above 20 micromolar CP. The amount of amino acids released in the presence of W-7 and W-5 was significantly lower than the amount released in the presence of CP. Both W-5 and W-7 increased amino acid release but only W-7 inhibited auxin-induced growth. Calmodulin activity measured by phosphodiesterase activation did not differ significantly between auxin-treated and control coleoptile segments. These results suggest the possible involvement of calmodulin in auxin-induced coleoptile elongation.     

Inhibition of the synthesis of cell wall polysaccharides in oat coleoptile segments by jasmonic acid: Relevance to its growth inhibition

The inhibitory mode of action of jasmonic acid (JA) on the growth of etiolated oat (Avena sativa L. cv. Victory) coleoptile segments was studied in relation to the synthesis of cell wall polysaccharides using [¹⁴C]glucose. Exogenously applied JA significantly inhibited indoleacetic acid (IAA)-induced elongation of oat coleoptile segments and prevented the increase of the total amounts of cell wall polysaccharides in both the noncellulosic and cellulosic fractions during coleoptile growth. JA had no effect on neutral sugar compositions of hemicellulosic polysaccharides but substantially inhibited the IAA-stimulated incorporation of [¹⁴C]glucose into noncellulosic and cellulosic polysaccharides. JA-induced inhibition of growth was completely prevented by pretreating segments with 30 mm sucrose for 4 h before the addition of IAA. The endogenous levels of UDP-sugars, which are key intermediates for the synthesis of cell wall polysaccharides, were not reduced significantly by JA. Although these observations suggest that the inhibitory mode of action of JA associated with the growth of oat coleoptile segments is relevant to sugar metabolism during cell wall polysaccharide synthesis, the precise site of inhibition remains to be investigated.Abbreviations JA jasmonic acid - ABA abscisic acid - IAA indoleacetic acid - T ₀ minimum stress relaxation time - TFA trifluoroacetic acid - TCA trichloroacetic acid - HPLC high-performance liquid chromatography - EtOAc ethyl acetate - TLC thin-layer chromatography - JA-Me methyl jasmonate - GLC-SIM gas-liquid chromatography-selected ion monitoring     

Role of chloride ions in the promotion of auxin-induced growth of maize coleoptile segments

Background and Aims

The mechanism of auxin action on ion transport in growing cells has not been determined in detail. In particular, little is known about the role of chloride in the auxin-induced growth of coleoptile cells. Moreover, the data that do exist in the literature are controversial. This study describes experiments that were carried out with maize (Zea mays) coleoptile segments, this being a classical model system for studies of plant cell elongation growth.

Methods

Growth kinetics or growth and pH changes were recorded in maize coleoptiles using two independent measuring systems. The growth rate of the segments was measured simultaneously with medium pH changes. Membrane potential changes in parenchymal cells of the segments were also determined for chosen variants. The question of whether anion transport is involved in auxin-induced growth of maize coleoptile segments was primarily studied using anion channel blockers [anthracene-9-carboxylic acid (A-9-C) and 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS)]. In addition, experiments in which KCl was replaced by KNO₃ were also performed.

Key Results

Both anion channel blockers, added at 0·1 mm, diminished indole-3-acetic acid (IAA)-induced elongation growth by ∼30 %. Medium pH changes measured simultaneously with growth indicated that while DIDS stopped IAA-induced proton extrusion, A-9-C diminished it by only 50 %. Addition of A-9-C to medium containing 1 mm KCl did not affect the characteristic kinetics of IAA-induced membrane potential changes, while in the presence of 10 mm KCl the channel blocker stopped IAA-induced membrane hyperpolarization. Replacement of KCl with KNO₃ significantly decreased IAA-induced growth and inhibited proton extrusion. In contrast to the KCl concentration, the concentration of KNO₃ did not affect the growth-stimulatory effect of IAA. For comparison, the effects of the cation channel blocker tetraethylammonium chloride (TEA-Cl) on IAA-induced growth and proton extrusion were also determined. TEA-Cl, added 1 h before IAA, caused reduction of growth by 49·9 % and inhibition of proton extrusion.

Conclusions

These results suggest that Cl^– plays a role in the IAA-induced growth of maize coleoptile segments. A possible mechanism for Cl^– uptake during IAA-induced growth is proposed in which uptake of K⁺ and Cl^– ions in concert with IAA-induced plasma membrane H⁺-ATPase activity changes the membrane potential to a value needed for turgor adjustment during the growth of maize coleoptile cells.     

Timing of the Auxin Response in Coleoptiles and Its Implications Regarding Auxin Action

The timing of the auxin response was followed in oat and corn coleoptile tissue by a sensitive optical method in which the elongation of about a dozen coleoptile segments was recorded automatically. The response possesses a latent period of about 10 min at 23°C, which is extended by low concentrations of KCN or by reducing the temperature, but is not extended by pretreatments with actinomycin D, puromycin, or cycloheximide at concentrations that partially inhibit the elongation response. Analysis of the data indicates that auxin probably does not act on the elongation of these tissues by promoting the synthesis of informational RNA or of enzymatic protein. Not excluded is the possibility that auxin acts at the translational level to induce synthesis of a structural protein, such as cell wall protein or membrane protein. While the data do not provide direct support for this hypothesis, the speed with which cycloheximide inhibits elongation suggests that continual protein synthesis may be important in the mechanism of cell wall expansion.     

Abscisic Acid localization and metabolism in barley aleurone layers

Aleurone layers of Hordeum vulgare, cv. `Himalaya' took up [¹⁴C]-abscisic acid (ABA) when incubated for various times. Radioactivity accumulated with time in a low speed, DNA-containing pellet accounting for 1.6 to 2.3% of the radioactivity recovered in subcellular fractions at 18 hours. Thin layer chromatography of ethanolic or methanolic extracts of the cytosol, which contained greater than 95% of the radioactivity taken up by layers, revealed that labeled ABA was metabolized to phaseic acid (PA) and 4′-dihydrophaseic acid (DPA) and three polar metabolites Mx₁, Mx₂, and Mx₃. ABA was not metabolized by endosperm, incubated under conditions used for layers, indicating that metabolism was tissue-specific. Layers metabolized [³H]DPA to Mx₁ and Mx₂. ABA, PA, and DPA-methyl ester and epi-DPA-methyl ester inhibited synthesis of α-amylase by layers incubated for either 37 or 48 hours. These layers converted the methyl DPA and epi-methyl-DPA esters to their respective acids. DPA did not inhibit Lactuca sativa germination or root and coleoptile elongation of germinating Hordeum vulgare seeds, or coleoptile elongation of germinating Zea mays seeds.     

Metabolic Requirement of Cucurbita pepo for Boron

Lateral roots of intact summer squash seedlings (Cucurbita pepo L.) were used to quantify the effects of boron deficiency on DNA synthesis, protein synthesis, and respiration. The temporal relationship between changes in these metabolic activities and the cessation of root elongation caused by boron deprivation was determined. Transferring 5-day-old squash seedlings to a hydroponic culture medium without boron for 6 hours resulted in a 62% reduction in net root elongation and a 30% decrease in the incorporation of [³H]thymidine into DNA by root tips (apical 5-millimeter segments). At this time, root tips from both boron-deficient and boron-sufficient plants exhibited nearly identical rates of incorporation of [¹⁴C]leucine into protein and respiration as measured by O₂ consumption. After an additional 6 hours of boron deprivation, root elongation had nearly ceased. Concomitantly, DNA synthesis in root apices was 66% less than in the boron-sufficient control plants and protein synthesis was reduced 43%. O₂ consumption remained the same for both treatments. The decline and eventual cessation of root elongation correlated temporally with the decrease in DNA synthesis, but preceded changes in protein synthesis and respiration. These results suggest that boron is required for continued DNA synthesis and cell division in root meristems.     

Studies on growth regulators. I. Improved Avena coleoptile elongation test for auxin

A modified procedure is presented for the bioassay of auxin using Avena coleoptile segments. The modifications introduced result in a substantial improvement of the commonly used coleoptile elongation tests.     

Jasmonic Acid Inhibits the IAA-Induced Elongation of Oat Coleoptile Segments: a Possible Mechanism Involving the Metabolism of Cell Wall Polysaccharides

The effects of jasmonic acid (JA) on the IAA-induced elongationof segments of etiolated oat (Avena sativa L. cv. Victory) coleoptileswere studied. Exogenously applied JA substantially inhibitedIAA-induced elongation of oat coleoptile segments. The inhibitionof the growth of oat coleoptile segments due to JA appeared2 h after the application of JA with IAA. JA did not affectthe consumption of oxygen by the segments, the osmolarity ofthe cell sap or the IAA-induced loosening of cell walls, whichwas recognized as a decrease in the minimum stress-relaxationtime (T₀). JA was extremely effective in preventing increasesin the amount of the cell wall polysaccharides in both the non-cellulosicfraction and the cellulosic fraction during coleoptile growthin the presence and in the absence of IAA. Inhibition of thegrowth of oat coleoptile segments induced by JA was partiallyreversed by the simultaneous addition of sucrose to the testsolution. From these results, it appears that JA inhibits IAA-inducedelongation of oat coleoptile segments by interfering with someaspects of sugar metabolism that are related to the degradationand/or the synthesis of cell wall polysaccharides. (Received March 15, 1994; Accepted August 2, 1994)     

Relationships between phytochrome state and photosensitive growth of Avena coleoptile segments

Using various photostationary state light sources to obtain reproducible phytochrome conversion of from 5 to 88% P_FR, assayed by 2 wavelength in vivo spectrophotometry, relationships between initial percent P_FR and elongation of apical Avena coleoptile segments over the succeeding 20 hours in darkness were studied. With material grown in total darkness, all P_FR levels promote elongation, and maximal promotion requires roughly 50% P_FR. The promotion caused by an initial 5 minute red (88% P_FR) treatment at hour 0 is partially reversible at hour 5 by sources forming less than 48% P_FR, but totally irreversible at hour 8, though less than 50% of the growth has been accomplished by this time. Direct photometric assays at hour 5 indicate a phytochrome state of roughly 45% P_FR, consistent with the reversal data. At hour 8, however, 11 to 22% of the phytochrome still assays as P_FR, an inconsistency suggesting simply that the elongation process has proceeded beyond photochemical control. Thus, in contrast with results previously reported for Pisum and Phaseolus, there is no contradiction between photometric and physiological assays of phytochrome state in Avena coleoptile segments.

Attempts to expand this study by using segments from seedlings pretreated with red light showed that such pretreatment as little as 1 to 2 hours before drastically reduces subsequent elongation and photoresponse on the medium employed. This decline in growth potential can be halted at any time before its completion by either excision of the segment or far-red treatment of the intact seedling.

     

In vivo evidence for the involvement of phospholipase A and protein kinase in the signal transduction pathway for auxin-induced corn coleoptile elongation

Auxin-induced elongation of com coleoptiles is accompanied by cell wall acidification, which depends upon H⁺-pump activity. We tested the hypothesis that phospholipase A and a protein kinase are involved in the pathway of auxin signal transduction leading to H⁺ secretion, and elongation of corn coleoptiles. Initially, the pH of the bath solution at 50–100 μm from the surface of a coleoptile segment (pH_o) ranged between 4.8 and 6.6 when measured with an H⁺-sensitive microelectrode. Twenty or 50 μM lysophosphatidylcholine, 50 μM linolenic acid or 50 μM arachidonic acid induced a decline in pH_o by 0.3 to 2.1 units. The effect was blocked by 1 mM vanadate, suggesting that lysophosphatidylcholine or linolenic acid induced acidification of the apoplast by activating the H⁺-pump. Lysophosphatidylcholine and linolenic acid also accelerated the elongation rate of the coleoptiles. While linolenic acid and arachidonic acid, highly unsaturated fatty acids, promoted pH_o decrease and coleoptile elongation, linoleic acid, oleic acid, and stearic acid, fatty acids with a lesser extent of unsaturation, had no such effects. The effects of lysophosphatidylcholine, linolenic acid, and arachidonic acid on H⁺ secretion were not additive to that of indoleacetic acid (IAA), suggesting that lysophospholipids, fatty acids and auxin use similar pathways for the activation of the H⁺-pump. The phospholipase A₂ inhibitors, aristolochic acid and manoalide, inhibited the IAA-induced pH_o decrease and coleoptile elongation. The general protein kinase inhibitors, H-7 or staurosporine, blocked the IAA- or lysophosphatidylcholine-induced decrease in pH_o. H-7 also inhibited the coleoptile elongation induced by IAA or lysophosphatidylcholine. These results support the hypothesis that phospholipase A is activated by auxin, and that the products of the enzyme, lysophospholipids and fatty acids, induce acidification of the apoplast by activating the H⁺-pump through a mechanism involving a protein kinase, which in turn promotes com coleoptile elongation.     

Induction of coleoptile elongation by carbon dioxide

The ability of CO₂ to induce elongation of Avena sativa coleoptile segments was examined with the use of a high resolution growth-recording device. CO₂-saturated water causes an 8- to 16-fold promotion in the rate of elongation within 1 minute. This elongation is insensitive to a variety of metabolic inhibitors that suppress auxin-induced elongation, and the CO₂ effect cannot be prevented by pretreatment with these inhibitors. Buffers of pH 3 to 4 also stimulate elongation quickly, and it seems that at least a major part of the action of CO₂ depends upon its ability to reduce pH. The rate of elongation of auxin-promoted segments can be further enhanced by treatment with CO₂ but not vice versa.     

Correlation Between Responses of Elongation of Wheat Coleoptile Segments to Abscisic Acid and Exogenous ATP

The time course of elongation and oxygen consumption of wheat coleoptile segments in 38μM ABA solution and 5 × 10-5 M 2,4-DNP solution were investigated. At the same time, effects of exogenous ATP (1 mM) on elongation and ABA response in wheat coleoptile segments were observed. Within the first hour of treatment, ABA and 2,4-DNP rapidly inhibited elongation in the coleoptile segments. Exogenous ATP stimulates elongation in coleoptile segments. When segments pretreated with ATP and then transfered into ABA solution, elongation in coleopfile segments were not inhibited. On the basis of the similarities of action pattern of ABA and 2,4-DNP and marked decrease of ABA inhibiting effect in presence of exogenous ATP, it is testified that uncoupling of respiration and oxidative phosphorylation is one of ABA important effects, so it is suggested that ABA inhibiting elongation of wheat coleoptile segments by decreasing tissue energy (ATP) levels necessary for synthetic reactions, including nucleic acid and protein, and associated with elongation.     

Red Light-inhibited Mesocotyl Elongation in Maize Seedlings: I. The Auxin Hypothesis

Red light-inhibited mesocotyl elongation, which occurs in intact Zea mays L. seedlings, was studied in excised segments which included the coleoptile (or parts therefrom) and apical centimeter of the mesocotyl. Experiments took into account, first, the ability of the segments to regenerate auxin supply sites, and, second, that auxin uptake can be greatly reduced if there is no cut surface, apical to the elongating cells, to act as a port of entry. In all cases, auxin completely reversed the inhibition of elongation by light. The results support the hypothesis that light regulates mesocotyl elongation by controlling auxin supply from the coleoptile. Sucrose concentration had no effect on auxin reversal of light-inhibited elongation, but relatively high concentrations of gibberellic acid (10 μm) could substitute for auxin in this system.     

The Effect of Gibberellin on Tryptophan Conversion and Elongation of the Avena Coleoptile

Gibberellic acid (GA₃) enhanced directly the release of ¹⁴CO₂ from tryptophan-1-^l4C by cell free preparations of Avena coleoptile tips. The rate of tryptophan metabolism in the presence of GA₃ was increased by approximately 100 per cent. The addition of auxin synthesis inhibitors to incubation flasks nullified the enhancement effect of GA₃ on elongation of the coleoptile tips. These studies implicate tryptamine as an intermediate in the formation of auxin from tryptophan. The possibility of GA₃-IAA interaction in the elongation processes was also investigated. Combination treatments of these growth-promoting substances did not induce a synergistic growth response by the coleoptile tissue.     

Inhibition of Low pH-induced Elongation in Avena Coleoptiles by Abscisic Acid

An angular position-sensing transducer was used to make continuous measurements of acid-induced elongation of Avena sativa coleoptile segments. Elongation rates at pH 4.5 (5 mm succinate buffer) were about 5-fold greater than those at pH 6.0. Buffered 0.1 mm abscisic acid produced a partial decrease of the growth rate. Pretreatments with abscisic acid buffered at pH 6.0 usually caused a further reduction of the elongation response when the coleoptile segments were subsequently placed in buffer at pH 4.5 containing abscisic acid. Abscisic acid did not completely prevent the pH effect in any of these experiments, and the brief latent period of the pH response was not affected by abscisic acid treatments. At pH 4.5, where the inhibitory effect of ABA was maximum, low pH-induced elongation was also inhibited by KCN and HgCl₂. These results suggest that pH-(4.5) induced elongation in this system may be dependent on some metabolic processes and that abscisic acid-induced inhibition of this elongation may involve an interaction with these processes.     

Cell wall development in maize coleoptiles

The physical bases for enhancement of growth rates induced by auxin involve changes in cell wall structure. Changes in the chemical composition of the primary walls during maize (Zea mays L. cv WF9 × Bear 38) coleoptile development were examined to provide a framework to study the nature of auxin action. This report documents that the primary walls of maize cells vary markedly depending on developmental state; polymers synthesized and deposited in the primary wall during cell division are substantially different from those formed during cell elongation.

The embryonal coleoptile wall is comprised of mostly glucuronoarabinoxylan (GAX), xyloglucan, and polymers enriched in 5-arabinosyl linkages. During development, both GAX and xyloglucan are synthesized, but the 5-arabinosyls are not. Rapid coleoptile elongation is accompanied by synthesis of a mixed-linked glucan that is nearly absent from the embryonal wall. A GAX highly substituted with mostly terminal arabinofuranosyl units is also synthesized during elongation and, based on pulse-chase studies, exhibits turnover possibly to xylans with less substitution via loss of the arabinosyl and glucuronosyl linkages.

     

Effects of the steroidal alkaloid tomatine in auxin bioassays and its interaction with indole-3-acetic acid

Summary The steroidal alkaloid tomatine did not enhance elongation of oat coleoptile and first internode sections, or of wheat coleoptile sections. Higher concentrations of the alkaloid inhibited elongation and interacted antagonistically with IAA. Although 10^-4 M tomatine alone did not influence elongation of oat coleoptile sections, it did reduce growth response to exogenous IAA. Tomatine concentrations less than 10^-4 M did not influence response to IAA. The auxin activity of tomatine, reported by Vendrig, was therefore not confirmed.