Effect of temperature on the dose–response curves for auxin-induced elongation growth in maize coleoptile segments

The dose–response curves for IAA-induced growth in maize coleoptile segments were studied as a function of time and temperature. In addition, the kinetics of growth rate responses at some auxin concentrations and temperatures was also compared. It was found that the dose–response curves for IAA-induced elongation growth were, independently of time and temperature, bell-shaped with an optimal concentration at 10⁻⁵ M IAA. The kinetics of IAA-induced growth rate responses depended on IAA concentration and temperature, and could be separated into two phases (biphasic reaction). The first phase (very rapid) was followed by a long lasting one (second phase), which began about 30 min after auxin addition. For coleoptile segments incubated at 30°C, the amplitudes of the first and second phase were significantly higher, when compared with 25°C, at all IAA concentrations studied. However, when coleoptile segments were incubated at 20°C, the elongation growth of coleoptile segments treated with suboptimal IAA concentrations was diminished, mainly as a result of both phases reduction. In conclusion, we propose that the shape of the dose–response curves for IAA-induced growth in maize coleoptile segments is connected with biphasic kinetic of growth rate response.     

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.     

Auxin Balance in the Avena Coleoptile

Coleoptiles of Avena possessed the capacity to degrade infiltrated indole-3-acetic acid (IAA). This activity decreased along the length of the coleoptile from apex to base on the bases of fresh weight, dry weight and protein; the apical 1 cm segment degraded more IAA than segments from other parts of the coleoptile. The naturally occurring inhibitor of the IAA oxidase activity increased in concentration up to 20 mm from the coleoptile apex; beyond, it decreased gradually towards the base. The spatial distribution of this inhibitor does not explain the gradient in IAA oxidase activity. Growth in length of the coleoptile and the IAA inactivating capacity of the apical 1 cm segment, increased 5- and 4,4-fold, respectively, between the ages of 70 and 130 h; but auxin secretion into agar platelets by the apical 2 mm of the coleoptile registered only a 2.7-fold increase. Deseeding and derooting the seedlings reduced the subsequent growth, diffusible auxin content and the IAA oxidase activity of the coleoptiles; derooting proved to be more deleterious than deseeding. A parallel reduction was evident in auxin content and IAA degrading activity following these treatments. Application of the cytokinin 6-benzylaminopurine (BAP) to coleoptiles of derooted seedlings failed to influence their capacity to degrade IAA. Nor was the activity of the aldehyde oxidase, which converts indole-3-acetaldehyde (IAAld) to IAA, affected by such treatment.     

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     

A comparison of the effects of IAA and 4-Cl-IAA on growth, proton secretion and membrane potential in maize coleoptile segments

The physiological activity of exogenous 4-Cl-IAA, as compared to IAA, was examined in maize coleoptile segments. It was found that in this model system 4-Cl-IAA is much more active in the stimulation of elongation than IAA. Simultaneous measurements of growth and external pH indicated that administration of either IAA or 4-Cl-IAA resulted in medium acidification. The kinetics of the pH changes, however, were faster after the addition of 4-Cl-IAA. In contrast to IAA, the coleoptile segments treated with chlorinated auxin were not able to increase medium pH after its initial drop. The re-addition of IAA after 5 h further enhanced growth over the next 2 h by 31%. By contrast, the re-addition of 4-Cl-IAA at the same time protocol as IAA did not cause an additional effect. The administration of 10 microM IAA induced in maize coleoptile cells a transient depolarization followed by a slow hyperpolarization of their membrane potential. In contrast to IAA, 4-Cl-IAA at 1 microM caused an immediate hyperpolarization of the membrane potential which, on average, was 2-fold greater than for IAA. The results reported here provide further evidence that 4-Cl-IAA is much more active, as compared to IAA, in stimulating the growth of maize coleoptile segments. Although it has not been directly demonstrated here, a plausible interpretation for the high 4-Cl-IAA activity is that, at least in part, it might be caused via a reduced metabolism of 4-Cl-IAA. Furthermore, for the first time, the data show that membrane potential responds to 4-Cl-IAA in a qualitatively different fashion than to IAA. These findings may, in turn, suggest a specific signal transduction pathway to 4-Cl-IAA in maize coleoptile cells.     

Rapid Inhibition of Auxin-induced Elongation of Avena Coleoptile Segments by Cordycepin

The effects of cordycepin (3'-deoxyadenosine), an RNA synthesis inhibitor, on auxin-induced elongation in Avena coleoptile segments were studied with a position-sensing transducer. Cordycepin rapidly inhibited auxin-stimulated growth in the coleoptile segments whether added before, at the same time as, or after, the 2 mum auxin treatment. Midcourse additions of 100, 50, and 25 mug/ml cordycepin inhibited auxin-promoted elongation in an average of 18, 22, and 35 minutes, respectively. Additions of cordycepin before or at the same time as the auxin treatment partially inhibited the magnitude of the subsequent auxin-promoted growth but did not appreciably alter the latent period of the auxin response. It was concluded that if cordycepin is inhibiting the synthesis of RNA required for growth, the decay time for this RNA may be considerably shorter than that suggested in the literature from actinomycin D experiments. Preliminary kinetic evidence indicated that cordycepin does not inhibit auxin-induced elongation by acting as a respiratory inhibitor. Studies in mung bean shoot mitochondria demonstrated that cordycepin has no effect on respiration, respiratory control, or ADP/oxygen ratios.     

Effects of red light on the growth of intact wheat and barley coleoptiles

The final lengths of intact dark-grown coleoptiles vary with species and cultivar. The growth distribution pattern in the apical 25-mm growing zone and the absolute amount of growth in each zone depend on the age and species of the coleoptile. A comparative study of several cultivars of wheat, Triticum vulgare, and barley, Hordeum vulgare, indicates that the growth distribution pattern in 30- to 38-mm coleoptiles varies with the species and cultivar. In barley, there are two patterns of growth distribution among the several cultivars, whereas in wheat, all cultivars exhibit a common zonal growth pattern. The total growth of coleoptiles, initially 30 to 38 mm in length, during a 24-hour dark incubation period is the same in dark-grown coleoptiles as in those irradiated with 3 minutes of red (660 nm) light prior to the incubation period. The growth distribution pattern in the growing zone of this 30- to 38-mm coleoptile is, however, altered by red light. Growth of the apical 5-mm zone is stimulated by red light and the zonal growth 5 to 10 mm below the apex is only slightly affected, whereas growth in the zones 10 to 15 to 20, and 20 to 25 mm below the apex is inhibited. This growth distribution pattern in irradiated coleoptiles changes as the coleoptile increases in length. The response of a zone following exposure to red light is dependent upon the age of the seedlings irradiated. The over-all effect of red light on growth of the intact coleoptile varies with the length of the coleoptile. In young seedling 20 to 29 mm in length, the cells of the coleoptile can compensate for the effects of red light, with the over-all growth of the dark-grown and irradiated coleoptile about the same. As the seedling grows older, the cells of the coleoptile can no longer make up for the effects of red light, and the over-all effect changes from compensation to pronounced inhibition.     

Interactive effects of temperature and heavy metals (Cd, Pb) on the elongation growth in maize coleoptiles

The effect of Cd and Pb on endogenous and IAA-induced elongation growth and medium pH of maize coleoptile segments incubated at 20, 25 and 30 °C was studied. It was found that the elongation of coleoptile segments and proton extrusion increased with the temperature and reached its maximum at 30 °C. For Cd, the maximal inhibition of endogenous and IAA-induced growth as well as medium acidification of coleoptile segments was observed at 25 °C. Meanwhile, Pb, irrespective of the temperature, diminished the growth of the segments by ca. 20%, increasing the acidification of the incubation medium. It was also found that in contrast to Cd, Pb accumulation in maize coleoptile segments did not correlate with temperature. The results suggest that the toxic effect of Cd on elongation growth of coleoptile segments is connected with the decrease of the PM H(+)-ATPase activity and probably with Cd-induced high acivity of IAA oxidase, whereas the effect of Pb did not depend on activity of any of the enzymes.     

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)     

Phytochrome Action in Oryza sativa L: IV. Red and Far Red Reversible Effect on the Production of Ethylene in Excised Coleoptiles

Excised apical segments of etiolated rice (Oryza sativa L.) coleoptiles produced ethylene. Increasing the number of cut sites per coleoptile increased the rate of ethylene formation. Ethylene produced by an etiolated-intact seedling in the dark was about a half of that by the excised coleoptile segment. Red light of low energy as well as of continuous irradiation inhibited the production of ethylene. The inhibition by a low energy dose of red light was partly relieved, if the red light was followed immediately by a small dose of far red light. The effect of red and far red light was repeatedly reversible, indicating that ethylene production was regulated by a phytochrome system. If the exposure to far red light was preceded by a period of darkness, this photoreversibility disappeared; 50% of the initial reversibility was lost within 5 hours. Applied ethylene (10 microliters per liter) significantly promoted the growth of intact coleoptiles of either totally etiolated or red light-treated seedlings, but had no effect on the excised apical segment of coleoptile.     

Cellular responses to naphthoquinones: juglone as a case study

The effects of juglone (JG) on the endogenous growth, growth in the presence of either indoleacetic acid (IAA) or fusicoccin (FC) and on proton extrusion were studied in maize coleoptile segments. In addition, membrane potential changes were also determined at chosen JG concentrations. It was found that JG, when added to the incubation medium, inhibited endogenous growth as well as growth in the presence of either IAA or FC. Simultaneous measurements of growth and external pH indicated that inhibition of either IAA-induced growth or proton extrusion by JG was a linear function of JG concentration. Addition of JG to the control medium caused depolarization of the membrane potential (E_m), value of which was dependent on JG concentration and time after its administration. Hyperpolarization of E_m induced by IAA was suppressed in the presence of JG. It was also found that for coleoptile segments initially preincubated with JG, although subsequently removed, addition of IAA was not effective in the stimulation of growth and medium acidification. Taken together, these results suggest that the mechanism by which JG inhibits the IAA-induced growth of maize coleoptile segments involves inhibition of PM H⁺-ATPase activity.     

Effect of temperature on growth,proton extrusion and membrane potential in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) coleoptile segments

The effects of temperature (5–45°C) on endogenous growth, growth in the presence of either indoleacetic acid (IAA) or fusicoccin (FC), and proton extrusion in maize coleoptile segments were studied. In addition, membrane potential changes at some temperatures were also determined. It was found that in this model system endogenous growth exhibits a clear maximum at 30°C, whereas growth in the presence of IAA and FC shows the maximum value in the range 30–35°C and 35–40°C, respectively. Simultaneous measurements of growth and external medium pH indicated that FC at stressful temperatures was not only much more active in the stimulation of growth, but was also more effective in acidifying the external medium than IAA. Also the addition of either IAA or FC to the bathing medium at 30 and 40°C did not change the kinetic characteristic of membrane potential changes observed for both substances at 25°C. However, the increased temperature significantly decreased IAA and FC-induced membrane hyperpolarization. IAA in the incubation medium, at 10°C, brought about additional membrane depolarization (apart from the one induced by low temperature). In contrast to IAA, FC at 10°C caused gradual repolarization of membrane potential, which correlated with both FC-induced growth and FC-induced proton extrusion. A plausible interpretation for temperature-induced changes in growth of maize coleoptile segments is that, at least in part, these changes were mediated via a PM H⁺-ATPase activity.     

Effects of UV-C radiation on extension growth, H+-extrusion and transmembrane electric potential in maize coleoptile segments

The effect of 253.7 nm ultraviolet radiation on elongation growth, medium acidification and changes in electric potential difference between vacuole and external medium in cells of maize ( Zea mays L.) coleoptile segments was investigated. It was found that irradiation with 390, 1170, 3900 and 5 850 J m⁻² UV-C (ultraviolet radiation 253.7 nm) inhibited elongation growth, whereas at 195 J m⁻² stimulation of growth was observed. The administration of IAA (10⁻⁵ M ) to the incubation medium of coleoptile segments partially abolished the inhibitory effect of UV-C. The pH of the incubation medium, measured simultaneously with growth, showed that the exposure of the segments to UV-C caused inhibition of H⁺-extrusion (or stimulation of H⁺ uptake). The presence of IAA (10⁻⁵ M ) in the incubation medium promoted (except after 5850 J m⁻² irradiation) H⁺-extrusion to a level comparable with that produced by IAA in non-irradiated segments. In UV-C irradiated segments the potential difference underwent significant alterations. Irradiation of coleoptile segments with 390 J m⁻² caused a transient depolarization, which was fully reversible within 30 min, while at higher doses depolarization was irreversible. The hyperpolarization of the membrane potential (MP) in cells of maize coleoptile induced by IAA was completely nullified by subsequent irradiation with UV-C. It is suggested that UV-C inhibited IAA-induced growth by a mechanism independent of cell wall acidification.     

Effect of thiosulphinates contained in garlic extract on growth, proton fluxes and membrane potential in maize (Zea mays L.) coleoptile segments

The effect of thiosulphinates contained in garlic extract (GE) on endogenous growth, growth in the presence of either indoleacetic acid (IAA) or fusicoccin (FC), and proton extrusion in maize coleoptile segments were studied. In addition, membrane potential changes at some GE dilutions and the protective effect of dithiothreitol (DTT) against GE toxicity were also determined. It was found that GE at almost all dilutions studied, when added to the incubation medium inhibited endogenous growth as well as growth in the presence of either IAA or FC. Simultaneous measurements of growth and external pH indicated that the administration of GE resulted in a complex change in the pH of the external medium; after an initial transient acidification, pH increased and reached the maximal value followed by a gradual decrease of medium pH. When IAA or FC was added after preincubation of the segments in the presence of GE the changes in medium pH were not significantly different from these obtained with GE only. If the coleoptile segments were first preincubated with GE and subsequently GE was removed, the addition of IAA induced strong growth and medium acidification. Dithiothreitol added together with GE neutralized the toxic effect of GE on growth of coleoptile segments incubated in the presence of IAA. The addition of GE to the control medium caused a depolarization of the membrane potential, the value of witch depended on GE dilution. These results indicate that the toxic effect of GE on growth of plant cells might be caused by disruption of the catalytic function of the plasma membrane H⁺-ATPase on formation of the disulfide bonds.     

Interactions of microtubule disorganizers, plant hormones, and red light in wheat coleoptile segment growth

Growth response of coleoptile segments excised from 3-day-old seedlings of wheat (Triticum vulgare cv. Baart) to gibberellic acid, indoleacetic acid, and 2,4-dichlorophenoxyacetic acid, to red light, and to several microtubule disorganizers depends on the initial position of the excised segment in the intact coleoptile. Red light, 660 nm, stimulates the growth of the apical cells, but inhibits markedly the growth of the cells in the basal region of the coleoptile. The effects of red light are independent of sucrose, gibberellic acid, indoleacetic acid, and 2,4-dichlorophenoxyacetic acid, even though these substances themselves markedly affect the growth of the coleoptile segments. Concentractions of the microtubule disorganizers, vinblastine sulfate, cupric chloride, urea, and colchicine, which do not alter significantly the growth of the dark control apical segments, substantially repress the promotive effects of red light or auxin on the increase in length of the apical cells of the coleoptile. This suggests that stimulation by red light and by auxin involves microtubule production. Microtubule disorganizers repress the growth of elongating cells of the coleoptile, yet on the other hand, auxin and irradiation do not alter significantly the response of basal cells to the microtubule disorganizing agents. We hypothesized that light and growth regulators induce polymerization of nonaggregated microtubule subunits, resulting in faster growth.     

Stimulatory effects of potassium iodide on auxin action in the coleoptile segments of maize (Zea mays)

Potassium iodide (KI) was found to stimulate IAA-induced elongation of coleoptile segments in maize (Zea mays L.). The promoting effects of KI on coleoptile elongation, which were optimal at 1 mM in the presence of IAA, did not occur as a result of better conservation of IAA in the incubation medium. In addition, KI did not affect fusicoccin- or epibrassinolide-induced elongation. Additionally, sodium iodide (NaI) induced similar stimulatory effects on IAA-induced elongation, however, potassium chloride (KCl) showed no effect, suggesting that iodide is the active component. KI also enhanced IAA-induced ethylene biosynthesis in maize coleoptile segments. Taken together, these results suggest the involvement of KI-sensitive step(s) in auxin action before effectors of the signal transduction pathway split to elongation growth and ethylene biosynthesis. In-yong Hwang and Soo Chul Chang contributed equally to this work.     

Auxin-induced extension, cell wall loosening and changes in the wall polysaccharide content of barley coleoptile segments

Contents of the cell wall and sugar pool and the response toexogenously applied auxin (cell extension and cell wall loosening)were investigated with barley coleoptile segments excised from4-, 5- and 6-day-old seedlings. The first two groups exhibiteda high capacity to grow in terms of the intact growth rate andwere responsive to auxin, while those excised from 6-day-oldseedlings had a low growth capacity. The cell wall of 4- and5-day-old coleoptile segments contained almost the same amountof noncellulosic wall components per unit length while the 6-day-oldones had a lesser amount. The sugar pool and -cellulose contentper unit length decreased as the coleoptile aged. Auxin-stimulatedextension was most marked in the 4-day-old coleoptile segments.Auxin caused quantitative changes in the cell wall componentsof 4-day-old coleoptiles and, to a lesser extent, of 5-day-oldcoleoptiles, i.e., an increase in the contents of xylose andarabinose, both of which are constituents of noncellulosic polysaccharidesof the cell wall, and of -cellulose and a decrease in the noncellulosicglucose content. Auxin caused very little change in the noncellulosicsugar content and -cellulose content of the cell wall from 6-day-oldcoleoptile segments. The auxin-induced change in mechanicalproperties of the cell wall was significant in 4- and 5-day-oldcoleoptiles but very small in 6-day-old ones. The results suggestedthat the content of noncellulosic wall components is closelyrelated to the intact growth and auxin responsiveness of barleycoleoptiles. (Received April 20, 1978; )     

The effects of electric field on the growth of intact seedlings and coleoptile segments ofZea mays L.

The experiments were carried out with 96-h-old intact maize seedlings and 10 mm long coleoptile segments cut 4 mm below the tip. The electric fields were applied longitudinally along the seedlings. The electric field (15 V) caused inhibition of the elongation growth of intact seedlings which was dependent on both the polarity and the duration of the applied voltage. The growth inhibition was greater when the tip of the shoot was positive relative to the roots. The electric field also caused inhibition of indole-3-acetic acid (IAA) and fusicoccin (FC) induced growth of maize coleoptile segments excised from electrically treated seedlings. IAA-induced growth of coleoptile segments was greater when the tip of the shoot was negative to the roots (not in the case of FC-treated segments and intact seedlings). It was suggested that apart from the changes induced by electric field in transport system of auxin the electric field affected also the activity of plasmalemma proton pump.     

beta-d-Glucan of Avena Coleoptile Cell Walls

A specific glucanase was used to liberate a noncellulosic beta-d-glucan from isolated cell walls of Avena sativa coleoptile tissue. Cell walls of this tissue contain as much as 7 to 9 mg of glucan/100 mg of dry wall. Because of the specific action pattern of the enzyme, a linkage sequence of.. 1 --> 4 Glc 1 --> 3 Glc 1 --> 4 Glc.. is indicated and the predominance of trisaccharide and tetrasaccharide as hydrolytic products suggests a rather regular repeating pattern in the polysaccharide. The trisaccharide and the tetrasaccharide are tentatively identified as 3-O-beta-cellobiosyl-d-glucose and 3-O-beta-cellotriosyl-d-glucose, respectively. Recovery of these oligosaccharides following glucanase treatment of native wall material was feasible only after wall-bound glucosidases were inactivated. In the absence of enzyme inactivation the released fragments were recovered as glucose. The beta-d-glucan was not extracted from walls by either hot water or protease treatment.Cell walls prepared from auxin-treated Avena coleoptile segments yielded less glucan than did segments incubated in buffer suggesting an auxin effect on the quantity of this wall component. No IAA-induced change in the ratio of the trisaccharide and tetrasaccharide could be detected, suggesting no shift in the 1,3 to 1,4 linkage ratio. While the enzyme acts directly on the beta-d-glucan, no elongation response was apparent when Avena sections were treated with the purified glucanase. The presence of the glucan was not associated with any wound response which could be attributed to the preparation of coleoptile segments. The relationship of glucan metabolism to auxin growth responses is discussed.