Effect of auxins on the auxin transport system in coleoptiles

Summary Within 20 min of its application, auxin enhances the transport processes that move auxin molecules through coleoptile tissue. The effect was observed after treatment with indoleacetic acid and -naphthaleneacetic acid, but not with growth-inactive auxin analogues.     

The electrical response of maize to auxins

The electrical response of Zea mays coleoptiles and suspension cultured cells to several growth-promoting auxins (IAA, IBA, 2,4-D, 2,4,5-T, 1-NAA) and some of their structural analogues (2,3-D, 2-NAA) has been tested. In coleoptile two typical electrical responses to IAA are observed: an immediated rapid depolarization, and a hyperpolarization following 7-10 minutes after the first external addition of IAA. Of the other tested compounds only 1-NAA significantly depolarized the cells, whereas all auxins as well as the analogues evoked delayed hyperpolarizations. In contrast, the suspension cells were not hyperpolarized by any of the tested compounds, but were strongly depolarized by IAA, 1-NAA, and to a lesser extent by 2-NAA. In these cells IAA and 1-NAA induced inwardly directed currents of positive charge which both saturated around 12 mA/m2. The strong pH-dependence together with the half-maximal currents 0.49 microM IAA and 0.76 microM 1-NAA point to a symport of the anions with at least 2H+. The delayed plasma membrane hyperpolarization is a different response, and seems to be initiated by the protonated auxin species. In accordance with the current literature, it is interpreted as consequence of a stimulated proton extrusion. The finding that all tested compounds evoked a hyperpolarization, makes this response unspecific. It is concluded that a stimulation of proton extrusion is a necessary, but not sufficient step to induce elongation growth.     

The electrical response ofAvena coleoptile cortex to auxins

Solute mobilities of 28 compounds in isolated cuticular membranes (CM) from Capsicum annuum L. fruit, Citrus aurantium L. and Pyrus communis L. leaves were studied using unilateral desorption from the outer surface. First-order rate constants of desorption (k^*), which are directly proportional to the diffusion coefficient in the waxy outer limiting skins of cuticles were measured. When log k^* was plotted vs. molar volumes of test compounds linear graphs were obtained. The y-intercepts of these graphs (k^*) represent the mobility of a hypothetical molecule having zero molar volume and the slopes of the graphs () represent the size selectivity of the barrier and are related to the free volume available for diffusion. Thus, solute mobilities in cuticles are composed of two independent terms which are subtractive. If k^* and are known, k^* can be estimated for any solute from its molar volume (V_x) using the equation log k^*=log k^* –V_x. These parameters were used to analyse the effects of plant species, extraction of cuticular waxes and molecular structure of solutes on solute mobilities in plant cuticles. For aliphatic solutes, k^* was a factor of 10 smaller than for cyclic compounds, while was 0.011 and 0.012, respectively. The k^*-values for CM of the three species were very similar, but was higher for bitter-orange CM (0.012) than for those of pepper fruits and pear leaves (0.009). This has the consequence that differences in solute mobilities (k^*) among cuticles from different plan species increase with increasing molar volumes of solutes. Our data and our analysis provide evidence that constituents of cuticular waxes are mobile, at least in the solid amorphous wax fraction, but mobility decreases rapidly with increasing molar volume. For instance, if amounts to 0.01, mobilities of wax monomers decrease by a factor of 10 for every increase in molar volume of 100 cm³ · mol^–1. Thus, hexadecanoic acid is quite mobile in the amorphous wax fraction of Citrus (k^*=1.5×10^–6·s^–1), but for dotriacontane having twice the molar volume, k^* was only 2.5×10^–9·s^–1, which is almost three orders of magnitude smaller. Wax esters have even higher molar volumes and their mobilities will be even smaller (about 4×10^–12·s^–1 for a C₄₈-ester). Since low chain mobilities are a prerequisite for low mobilities and permeabilities, the selective advantage of high-molecular-weight wax monomers in plant cuticular waxes becomes obvious. Extracting cuticular waxes from pear leaf CM increased solute mobilities by a factor of 182, but it had no effect on size selectivity. We interpret this result as evidence to the effect that cuticular waxes reduce mobility by increasing tortuosity of the diffusion path, rather than by decreasing the mean free path of diffusional jumps and jump frequencies of diffusants.Abbreviations CM cuticular membrane(s) - 2,4-D 2,4-dichloro-phenoxyacetic acid - LAB lactic acid buffer - MX polymer matrix membranes - UDOS unilateral desorption from the outer surface     

Gravitational compensation and the phototropic response of oat coleoptiles

Avena seedlings were germinated and grown while continuously rotated on the horizontal axis of a clinostat. The coleoptiles of these gravity-compensated plants were phototropically more responsive than those of plants rotated on a vertical axis. When the plants were compensated after unilateral irradiation, phototropic curvature of the shoot progressed for the next 6 hours, with the rate of curving decreasing about 3 hours after irradiation. The decrease in rate was less in the plants gravity-compensated before irradiation than in those vertically rotated. In the period 70 to 76 hours after planting, the growth rate of the compensated coleoptiles was significantly less than that of the vertically rotated seedlings. The greater phototropic curvature, the decreased growth rate, and the slower rate of straightening of the curved, compensated shoot can be correlated with several consequences of compensation: an increase in sensitivity to auxin, a lowering of auxin content in the coleoptile tip, and possibly, from an interaction between compensation and phototropic stimulation, an enhanced difference in auxin transport between the illuminated and shaded halves of the unilaterally irradiated shoot.

The phototropic response of the vertically rotated seedling was significantly different from that of the vertical stationary, indicating the importance of vertically rotated controls in clinostat experiments.

     

Lateral electrical potential following asymmetric auxin application to maize coleoptiles

Following asymmetric application of indoleacetic acid to maize (Zea mays L.) coleoptiles the early time course of changes in lateral electrical potential was externally monitored with static-drop electrodes. First, an early negative potential change of ca.-1 mV was measured at the surface on the side of a strong auxin application. This negative auxin effect ended after ca. 15 min and was followed by a strong and lasting auxin stimulation of a positive lateral potential up to +12 mV at the auxin-treated side. The initial auxin effect appeared to depend on the size of the step-up in auxin concentration.     

Rapid response of the plasma-membrane potential in oat coleoptiles to auxin and other weak acids

We have compared the effects of the auxin, indole-3-acetic acid (IAA) with that of other weak acids on the plasma-membrane potential of oat (Avena sativa L.) coleoptile cells. Cells treated with 1 M IAA at pH 6 depolarize 20–25 mV in 10–12 min, but they then repolarize, until by 20–25 min their potentials are about 25 mV more negative than the initial value. Similar concentrations of benzoic and butyric acids cause the initial depolarization, but not the subsequent hyperpolarization. The hyperpolarization is therefore specific to IAA. All the weak acids, including IAA, evoke a rapid hyperpolarization when their concentrations are raised to 10 mM. This result indicates that at high concentrations, the uptake of undissociated weak acids activates electrogenic proton pumping, most likely by lowering cytoplasmic pH. In contrast, the hyperpolarization observed with concentrations of IAA four orders of magnitude lower appears to be a specific hormonal effect. This specific, auxin-induced hyperpolarization occurs at the same time as the initiation of net proton secretion and supports the hypothesis that auxin initiates extension growth by increasing proton pumping.Abbreviations FC fusicoccin - IAA indole-3-acetic acid     

Rooting, growth and ethylene evolution of pea cuttings in response to chloroindole auxins

In pea cuttings ( Pisum sativum L. cv. Alaska) we measured shoot and root growth and ethylene production in response to 4-chloroindole-3-acetic acid (4-CI-IAA) or 4,6-dichloroindole-3-acetic acid (4,6-Cl₂-IAA). Leafy cuttings treated basally with either of the chlorinated auxins in high concentrations showed permanent epinasty, loss of apical growth and dominance resulting in the outgrowth of laterals from the lower-most axillary bud. The naturally occurring 4-CI-IAA was a better root promoter than the synthetic 4,6-Cl₂-IAA which inhibited rooting. Both chloroindole auxins induced very high ethylene evolution, which lasted much longer than the ethylene evolution after IAA treatment.     

Ethylene and carbon dioxide as mediators in the response of the bean hypocotyl hook to light and auxins

Summary Ethylene inhibits hook opening in the bean hypocotyl and at high concentrations induces closure of the hook. Indoleacetic acid and 2,4-dichlorophenoxyacetic acid, whose inhibitory effect on hook opening resembles that of ethylene, stimulate ethylene production from the hook tissue, and this ethylene production is physiologically active in inhibiting hook opening. It is concluded that the inhibition of opening by auxin is due at least in a major part to auxin-induced ethylene production by the hook tissue.Carbon dioxide promotes hook opening, apparently by antagonizing the action of endogenous ethylene. The concentration of respiratory CO₂ in the internal gas space of the hook tissue is high enough to play a role in the regulation of hook opening.Red light causes a decrease in ethylene production and an increase in CO₂ evolution from the hook tissue. These effects are partially reversible by far-red light. It is concluded that both ethylene and CO₂ serve as natural growth regulators which mediate the hypocotyl hook-opening response to light in bean seedlings.     

Mechanisms mediating the thermal response to morphine withdrawal in rats

Studies were undertaken to evaluate the role of peripheral adrenergic mechanisms and the adrenal gland in the thermal responses which accompany morphine withdrawal in the rat. Ovariectomized rats were addicted to morphine and subsequently withdrawn by administration of naloxone. This treatment resulted in a significant rise (5-6 degrees C) in tail skin temperature (TST) and fall in colonic temperature (2-4 degrees C). Systemic administration of clonidine (0.5 mg/kg) completely suppressed this surge in TST and significantly attenuated the fall in core temperature. Similar results were observed following the systemic administration of ST-91, another alpha 2-adrenergic agonist which does not cross the blood-brain barrier. Central administration of ST-91 (50 micrograms/5 microliters, icv) was also successful in attenuating these temperature changes in the morphine-dependent rat. Adrenalectomy and peripheral administration of propranolol (10 mg/kg sc) both resulted in a significant attenuation of the surge in TST and the fall in core temperature in the morphine-dependent rat which suggest some peripherally mediated event is necessary to produce the full skin temperature surge. Collectively, the data suggest a role for the adrenal gland and adrenergic receptors in producing the surge in TST in morphine-dependent rats. It also suggests that the blocking effects of the alpha 2-adrenergic agonist can be mediated both centrally and peripherally.     

Developmental changes in the secondary xylem ofAcer rubrum induced by various auxins and 2,3,5-tri-iodobenzoic acid

Summary TIBA has been applied laterally to actively growing stems of uprightAcer rubrum seedlings. The frequency of initiation of tracheary elements is reduced and a complete ring of tension wood is developed in the stem locally below the TIBA application site. Rings of tension wood were never formed above the TIBA treatment site. In regard to anatomy, lignin distribution and peroxidase activity, the tension wood fibers formed as a result of TIBA treatment are identical to those which can be induced by bending.In the region of the stem above the site of TIBA application there is a particularly strong alteration in the development of tracheary elements.Application of IAA, NAA, or 2,4-D to the TIBA treatment site suppresses the capacity of TIBA to induce the development of tension wood and at the same time generally increases the frequency of initiation of tracheary elements.The effect of auxin alone on theAcer rubrum system has been studied. The secondary xylem formed during treatment with auxins (especially 2,4-D and NAA) at the stated concentrations is generally characterized by large groups of tracheary elements with a conspicuous angular outline in transverse section.The evidence suggests that auxins are involved in the regulatory systems which bring about the orderly development of the secondary xylem in arborescent angiosperms.This material was included in a doctoral thesis submitted by P. R.Morey to the graduate school of Yale University, New Haven.     

Different affective response to opioid withdrawal in adolescent and adult mice

AimsDrug withdrawal is suggested to play a role in precipitating mood disorders in individuals with familial predisposition. Age-related differences in affective responses to withdrawal might explain the increased risk of mental illnesses when drug use begins during adolescence. Since there is a lack of animal research examining the effects of opioid withdrawal during adolescence, the present study examined whether there are age-related differences in affective responses to opioid withdrawal.Main methodsAdolescent and adult mice were injected with two different morphine regimens, namely low and high, which differed in the dosage. Three and nine days following discontinuation of morphine administration, immobility time in the forced swim test (FST) and locomotion (total distance traveled) were evaluated.Key findingsOn withdrawal day 3 (WD3), adolescent mice exhibited a decrease in immobility as compared to controls. No significant differences in immobility were observed on withdrawal day 9 (WD9). This effect on FST behaviors was not due to changes in overall motor activity, since no differences in locomotion were observed on either WD3 or WD9 in adolescent mice. In adults, no differences in either FST or locomotor behaviors were observed on WD3. As expected, on WD9, adult mice exhibited an increase in immobility and a decrease in locomotion.SignificanceThis study demonstrates age-dependent differences in both FST scores and locomotor behaviors during opioid withdrawal. FST behaviors are classically used to evaluate mood in rodents, thus this study suggests that opioid withdrawal might affect mood differentially across age.     

The phototropic response of Triticum aestivum coleoptiles under conditions of low gravity

Wheat (Triticum aestivum L. cv Broom) coleoptiles were stimulated by unilateral blue light pulses, the duration of which varied between 3 s and 30 min, under microgravity conditions on the IML-1 Spacelab mission and in ground controls. The stimuli covered first positive, indifferent and second positive response regions. When phototropic responses were observed under low-gravity conditions, slightly fewer seedlings responded compared with 1 g conditions. The latent period was similar in flight and 1g treatments (10–20 min), except for the response to 300,μmol m^?2 in the indifferent response region, where a positive response in flight plants followed a 130 min latent period, while no response was observed in 1 g plants. First positive responses at Og were slightly enhanced, both in magnitude and in duration, compared to the 1 g controls, but not to the extent predicted by clinostat studies. The response kinetics in the second positive region at 1g showed a plateau at 120 min, in contrast to the single maximum at Og. The fluence-response relationship was similar in both flight and ground controls. Only the responses to 4 and 6μmolm-2 showed significantly greater curvatures at 9g. This contrasts with previous clinostat studies, which reported substantial response enhancement at all fluence levels.     

Cyclic AMP response to various haemopoietic regulators

Diffusible inhibitors and stimulators are involved in the regulation of bone marrow pluripotent stem cell (CFU-S) proliferation. We have previously shown the existence of CFU-S inhibitors in foetal calf marrow and liver and have started their purification. The lack of a simple and time-saving test to determine the kinetic state of CFU-S and the activity of the inhibitors led us to explore the possibility of a biochemical proliferation marker that could be used for screening purpose. Since it was shown that cyclic AMP was implicated in the regulation of CFU-S proliferation, it was of interest to study the variations in cAMP levels after stimulation and inhibition of CFU-S entry into cycle. The results of in vitro experiments showed that the increase in cAMP levels observed in bone marrow cells after incubation with different haemopoietic stimulators was specific neither for bone marrow cells nor for the various haematopoietic regulators. In the in vivo experiments, an increased cAMP level was observed 8 hr after one injection of Ara-C at the time when CFU-S are recruited into S phase. However, no modification of cAMP levels has been observed after injection of CFU-S inhibitors in the Ara-C-treated mice. Although cAMP does not seem to be a suitable marker for testing the activity of inhibitory fractions during the purification process, this work has contributed to the study of CFU-S stimulators.     

Enhancement of phototropic response to a range of light doses in Triticum aestivum coleoptiles in clinostat-simulated microgravity

The phototropic dose-response relationship has been determined for Triticum aestivum cv. Broom coleoptiles growing on a purpose-built clinostat apparatus providing gravity compensation by rotation about a horizontal axis at 2 rev·min^-1. These data are compared with data sets obtained with the clinostat axis vertical and stationary, as a 1·g control, and rotating vertically to examine clinostat effects other than gravity compensation. Triticum at 1·g follows the wellestablished pattern of other cereal coleoptiles with a first positive curvature at low doses, followed by an indifferent response region, and a second positive response at progressively increasing doses. However, these response regions lie at higher dose levels than reported for Avena. There is no significant difference between the responses observed with the clinostat axis vertical in the rotating and stationary modes, but gravity compensation by horizontal rotation increases the magnitude of first and second positive curvatures some threefold at 100 min after stimulation. The indifferent response is replaced by a significant curvature towards the light source, but remains apparent as a reduced curvature response at these dose levels.     

Interaction of gravi- and phototropic stimulation in the response of maize (Zea mays L.) coleoptiles

The influence of gravitropic stimulation upon blue-light-induced first positive phototropism for stimulations in the same (light source and center of gravity opposite to each other) and in opposing directions was investigated in maize cole-optiles by measuring fluence-response patterns. As a result of gravitropic counterstimulation, phototropic bending was transient with maximum curvature occurring 100 min after stimulation. On a horizontal clinostat, however, the seedlings curved for 20 h. Gravistimulation in the opposite direction acted additively upon blue-light curvature. Gravistimulation in the same direction as phototropic stimulation produced a complex behaviour deviating from simple additivity. This pattern can be explained by a gravitropically mediated sensitization of the phototropic reaction, an optimal dependence of differential growth on the sum of photo-and gravistimulation, and blue-light-induced inhibition of gravitropic curvature at high fluences. These findings indicate that several steps of photo-and gravitransduction are separate. Preirradiation with red light desensitized the system independently of applied gravity-treatment, indicating that the site of red-light interaction is common to both transduction chains.Abbreviations BL blue light - G⁺ stimulation by light and gravity in the same direction (i.e. light source and center of gravity opposite to each other) - G^- stimulation by light and gravity in opposing directions     

The effect of brefeldin A on the redistribution of osmiophilic particles and the gravitropic response of rye coleoptiles

Summary The occurrence of IAA-inducible osmiophilic particles (OPs) in the periplasmic space of epidermal cells in the upper and lower flank (UF, LF) of gravistimulated rye coleoptile segments was analyzed employing brefeldin A (BFA) as an inhibitor of secretion at the plasma membrane. A 2 h horizontal gravistimulation of untreated samples caused a duplication of OPs in the periplasmic space of epidermal cells at the growth-inhibited UF as compared to the LF of upward bending coleoptile segments. In contrast to this, the number of OPs within the cytoplasm close to the plasma membrane of epidermal cells was similar at both flanks. BFA caused an inhibition of graviresponsive growth and prevented the occurrence of OPs in the periplasmic space of the epidermal cells of the UF and the LF. Likewise, growth of vertically oriented coleoptile segments was inhibited by BFA. Growth inhibition of both gravistimulated and control segments was accompanied by a twofold increase of the occurrence of cytoplasmic OPs. The results illustrate that the occurrence of OPs within the periplasmic space of the epidermal cells depends on secretion processes. Furthermore they provide evidence that their increased occurrence in the growth-inhibited UF during gravistimulation is due to their inhibited infiltration into the cell walls. We suggest that thereby wall loosening is temporarily prevented.Abbreviations BFA brefeldin A - LF lower flank - OP osmiophilic particle - UF upper flank Dedicated to Professor Dr. Dieter Klämbt on the occasion of his 65th birthday