Pulse-induced phototropisms in oat and maize coleoptiles

Phototropisms induced by a pulse (1-30 seconds) of blue light in red-light-grown coleoptiles of oats (Avena sativa L.) and maize (Zea mays L.) were investigated in terms of fluence-response relationships and time courses. Phototropic stimulation was made by a laser beam (457.9 nanometers), allowing application of high-fluence pulses. The phototropic fluence-response curves for oats and maize revealed two peaks in the positive range, thus indicating the occurrence of two separable pulse-induced positive responses. The response at low fluences corresponded to the `first positive curvature.' The response at high fluences was very small in oats, but was large in maize. Reciprocity was valid in this high-fluence response (tested only for maize), indicating that it is distinct from the so-called `second positive curvature.' In oats, the trough between the two positive responses fell into the negative range. This negative response, corresponding to the `first negative curvature,' showed time courses distinct from those of `first positive curvature:' the negative response was induced after a longer time lag and developed with a more gradual increase of the rate of bending. The maximal rate of the negative response was as high as one-half of that of first positive curvature. In maize, the trough between the two responses was in the positive range, and the time-course result revealed no apparent response counteracting the positive responses. Physiological and ecological implications of the pulse-induced phototropisms are discussed.     

Electrical evidence for different mechanisms of uptake for basic, neutral, and acidic amino acids in oat coleoptiles

The application of neutral or acidic amino acids to oat coleptiles induced transient depolarizations of the membrane potentials. The depolarizations are considered to reflect H⁺ -amino acid co-transport, and the spontaneous repolarizations are believed to be caused by subsequent electrogenic H⁺ extrusion. The basic amino acids depolarized the cell membrane strongly, but the repolarizations were weak or absent. The depolarizations induced by the basic amino acids were weakly sensitive to manipulations of the extracellular and intracellular pH. The depolarizations induced by the other amino acids, in contrast, were more strongly affected by the pH changes. Several amino acids induced distinct but diminished depolarizations in the presence of 2,4-dinitrophenol or cyanide, but the repolarizations were generally eliminated. These experiments support the co-transport theory but suggest somewhat different mechanisms for the transport of the neutral, acidic, and basic amino acids. We suggest that the neutral amino acids are co-transported with a single H⁺ and that accumulation depends upon both the ΔpH and the membrane potential components of the proton motive force. The acidic amino acids appear to be accumulated by a similar mechanism except that the transport of each molecule may be associated with a cation in addition to a single proton. The permanently protonated basic amino acids appear not to be co-transported with an additional proton. Accumulation would depend only on the membrane potential component of the proton motive force.     

Ethanol sensitivity of rice and oat coleoptiles

The ability to avoid the ethanol-induced injury was evaluated in rice ( Oryza sativa L.) and oat ( Avena sativa L.) coleoptiles. The growth of the rice and oat coleoptiles was inhibited by ethanol exogenously applied at concentrations greater than 200 and 30 m M , respectively. At 300 m M ethanol, oat coleoptiles were brown and flaccid but rice coleoptiles did not show any visible symptoms of toxicity. The acetaldehyde level in rice and oat coleoptiles was increased by exogenously applied ethanol and the increases were greater in oat than in rice coleoptiles under aerobic and anaerobic conditions. At 300 m M ethanol, the acetaldehyde concentrations in the rice and oat coleoptiles were 46 and 87 nmol g⁻¹ FW under aerobic conditions, respectively, and 52 and 124 nmol g⁻¹ FW under anaerobic conditions, respectively. The activity of alcohol dehydrogenase (ADH; EC 1.1.1.1) in the direction of ethanol to acetaldehyde was greater in oat than in rice coleoptiles and ADH protein in oat coleoptiles was more induced by exogenously applied ethanol than that in rice coleoptiles. These results suggest that in vivo conversion rate of ethanol to acetaldehyde by ADH is lower in rice than oat coleoptiles, which may be one of the reasons that ethanol sensitivity of rice is much lower than that of oat coleoptiles. The great ability of rice to avoid the ethanol-induced injuries may contribute its anoxia tolerance when glycolysis and ethanolic fermentation replace the Krebs cycle as the main source of energy under anaerobic conditions.     

Derepression of amino Acid-h cotransport in developing soybean embryos

The uptake of the unnatural amino acid α-aminoisobutyric acid (AIB) and glutamine by developing soybean (Glycine max Merr. cv Chippewa 64) embryos was investigated. In freshly excised embryos, the accumulation ratio (cytoplasmic concentration/external concentration) of AIB did not exceed 1.0. After an 18-hour preincubation in nitrogen-free medium the accumulation ratio of AIB exceeded 4.5 at an external AIB concentration of 10 micromolar. This indicates the derepression of an active amino acid uptake mechanism operative at low external amino acid concentration. The presence of sucrose, NH₄NO₃, or glutamine during a 21-hour preincubation prior to measuring glutamine uptake inhibited the enhancement of uptake by 43%, 51%, and 96%, respectively. The time course of the decline in free amino acids and the time course of enhancement of amino acid uptake was not consistent with enhanced uptake resulting from relief of transinhibition, but suggested instead the derepression of synthesis of new carriers. The time course of enhancement of amino acid uptake was paralleled by an increase in glutamine-induced depolarization of the membrane potential. The kinetics of glutamine uptake indicated the presence of a saturable and a nonsaturable component of uptake. The saturable component of uptake is attributed to a mechanism of amino acid-H⁺ cotransport which is derepressed by nitrogen and/or carbon starvation. At physiological concentrations of amino acids, uptake through the saturable system in freshly excised embryos is negligible. Thus, uptake through the nonsaturable system is of primary importance in the nitrogen nutrition of developing soybean embryos.     

Evidence from studies with acifluorfen for participation of a flavin-cytochrome complex in blue light photoreception for phototropism of oat coleoptiles

The diphenyl ether acifluorfen enhances the blue light-induced absorbance change in Triton X100-solubilized crude membrane preparations from etiolated oat (Avena sativa L. cv. Lodi) coleoptiles. Enhancement of the spectral change is correlated with a change in rate of dark reoxidation of a b-type cytochrome. Similar, although smaller, enhancement was obtained with oxyfluorfen, nitrofen, and bifenox. Light-minus-dark difference spectra in the presence and absence of acifluorfen, and the dithionite-reduced-minus oxidized difference spectrum indicate that acifluorfen is acting specifically at a blue light-sensitive cytochrome-flavin complex. Sodium azide, a flavin inhibitor, decreases the light-induced absorbance change significantly, but does not affect the dark reoxidation of the cytochrome. Hence, it is acting on the light reaction, suggesting that the photoreceptor itself is a flavin. Acifluorfen sensitizes phototropism in dark-grown oat seedlings such that the first positive response occurs with blue light fluences as little as one-third of those required to elicit the same response in seedlings grown in the absence of the herbicide. Both this increase in sensitivity to light and the enhancement of the light-induced cytochrome reduction vary with the applied acifluorfen concentration in a similar manner. The herbicide is without effect either on elongation or on the geotropic response of dark-grown oat seedlings, indicating that acifluorfen is acting specifically close to, or at the photoreceptor end of, the stimulus-response chain. It seems likely that the flavin-cytochrome complex serves to transduce the light signal into curvature in phototropism in oats, with the flavin moiety itself serving as the photoreceptor.     

Role of cell-wall-degrading enzymes in cell-wall loosening in oat coleoptiles

Summary Effects of auxin (indole-3-acetic acid), fungal -1,3-glucanase and pectin methylesterase on expansion and on cell wall extensibility, measured by the extensometer technique, of oat coleoptile segments were studied. Pretreatment with these substances for less than 30 min promoted tissue expansion remarkably. Under osmotic stress by 0.25 M mannitol, which prevented uptake of water by the cells, auxin increased DE but not DP in 30- and 60-min incubations. -1,3-Glucanase or -1,3-glucanase plus pectin methylesterase also increased only DE under the same conditions. A role of cell-wall-degrading enzymes in initiating cell expansion is therefore suggested.     

Intracellular localisation of phytochrome in oat coleoptiles by electron microscopy

We have analysed the intracellular localisation of phytochrome in oat coleoptile cells by electron microscopy and confirm and extend light-microscopical findings of previous authors. We used indirect immuno-labeling with polyclonal antibodies against 60-KDa phytochrome from etiolated oat seedlings, and a gold-coupled second antibody, on ultrathin sections of LR-white-embedded material. In dark-grown seedlings, phytochrome-labeling is distributed diffusely throughout the cytoplasm. Organelles and membranes are not labeled. After photoconversion of the red-absorbing form of phytochrome to the far-red absorbing form (Pfr) (5-min red light; 660 nm), the label is sequestered uniquely in electron-dense areas within the cytoplasm. These areas are irregularly shaped, are often located in the vicinity of the vacuole, are not surrounded by a membrane, exclude cellular organelles and ribosomes and are not found in dark-grown material; an immediate 5-min farred light pulse after the red light does not cause these structures to disappear. After a dark period of 3–4 h following red-light irradiation, these electron-dense structures disappear together with any specific labeling. We suggest a Pfr-induced aggregation of an unknown, phytochrome-binding protein or proteins.Abbreviations Pr and Pfr phytochrome in its red and far-red absorbing form, respectively     

Intracellular localisation of phytochrome in oat coleoptiles by electron microscopy

The intracellular localisation of phytochrome in oat (Avena sativa L. cv. Garry Oat) coleoptiles was analysed by electron microscopy. Serial ultrathin sections of resin-embedded material were indirectly immunolabeled with polyclonal antibodies against phytochrome together with a gold-coupled second antibody. The limits of detectability of sequestered areas of phytochrome (SAPs) were analysed as a function of light pretreatments and amounts of the far-red absorbing form of phytochrome (Pfr) established. In 5-d-old dark-grownAvena coleoptiles SAPs were not detectable if less than 13 units of Pfr — compared with 100 units total phytochrome of 5-d-old dark-grown seedlings — were established by a red light pulse. In other sets of experiments, seedlings were preirradiated either with a non-saturating red light pulse to allow destruction to occur or with a saturating red followed by a far-red light pulse to induce first SAP formation and then its disaggregation. These preirradiations resulted in an increase of the limit of detectability of SAP formation after a second red light pulse to 38–41 and 19–23 units Pfr, respectively. We conclude that with respect to Pfr-induced SAP formation an adaptation process exists and that our data indicate that SAP formation is not a simple self-aggregation of newly formed Pfr.Abbreviations FR far-red light - Pfr, Pr far-red-absorbing and red-absorbing forms of phytochrome, respectively - Plot total phytochrome (Pfr + Pr) - R red light - SAP sequestered areas of phytochrome This work was supported by Deutsche Forschungsgemeinschaft (SFB 206). The competent technical assistance of Karin Fischer is gratefully acknowledged.     

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.

     

Changes in ion fluxes during phototropic bending of etiolated oat coleoptiles

BACKGROUND AND AIMS: This work has been conducted to assist theoretical modelling of the different stages of the blue light (BL)-induced phototropic signalling pathway and ion transport activity across plant membranes. Ion fluxes (Ca(2+), H(+), K(+) and Cl(-)) in etiolated oat coleoptiles have been measured continuously before and during unilateral BL exposure. METHODS: Changes in ion fluxes at the illuminated (light) and shadowed (dark) sides of etiolated oat coleoptiles (Avena sativa) were studied using a non-invasive ion-selective microelectrode technique (MIFE). The bending response was also measured continuously, and correlations between the changes in various ion fluxes and bending response have been investigated. For each ion the difference (Delta) between the magnitudes of flux at the light and dark sides of the coleoptile was calculated. KEY RESULTS: Plants that demonstrated a phototropic bending response also demonstrated Ca(2+) influx into the light side approximately 20 min after the start of BL exposure. This is regarded as part of the perception and transduction stages of the BL-induced signal cascade. The first 10 min of bending were associated with substantial influx of H(+), K(+) and Cl(-) into the light (concave) side of the coleoptiles. CONCLUSIONS: The data suggest that Ca(2+) participates in the signalling stage of the BL-induced phototropism, whereas the phototropic bending response is linked to changes in the transport of H(+), K(+) and Cl(-).     

Phototropism and photoinhibition of basipolar transport of auxin in oat coleoptiles

We have proposed that the lateral inequality of auxin associated with the phototropic response is a consequence of a light-induced impairment of basipolar transport of the hormone. If this is so, the dose-response curve for photoinhibition of auxin transport should resemble that for phototropism. Further, the wavelength dependencies of the transport and tropic response should also be similar. Oat coleopilles were irradiated equilaterally with white light and with broad- and narrow-spectral bands of blue light. Indoleacetic acid 2-¹⁴C was then applied apically to the intact coleoptile. Irradiation by the 3 sources inhibited basipolar transport of the auxin. The photoinhibition of transport increases with exposure, reaches a maximum at radiant energies depending on the source used, and then decreases. The dose response for transport inhibition matches that for phototropism. A correspondence in the spectral response of the 2 phenomena is also found for coleoptiles exposed to first positive energies at various wavelengths from 340 to 730 nm. We interpret these correlations as support for the hypothesis that phototropism is mediated by a photoinhibition of basipolar transport of auxin.     

Anoxia tolerance and ethanol sensitivity of rice and oat coleoptiles

Anoxia tolerance and ethanol sensitivity of rice (Oryza sativa L.) and oat (Avena sativa L.) seedlings were evaluated to clarify their growth habit in anoxia. Anoxic stress inhibited elongation and dry weight gain of coleoptiles of the oat and rice seedlings; however, the inhibition of the oat coleoptiles was much greater than that of the rice coleoptiles. Anoxic stress increased endogenous ethanol concentration and alcohol dehydrogenase activity in oat and rice coleoptiles and their increases in the rice coleoptiles were much greater than those in the oat coleoptiles. At concentrations greater than 30 mM and 300 mM, exogenously applied ethanol inhibited the elongation and weight gain for the oat and the rice coleoptiles, respectively, and the inhibition was increased with increasing ethanol concentrations with marked inhibition being achieved on the oat coleoptiles. These results suggest that anoxia tolerance and induction of ethanolic fermentation in anoxia may be greater in rice than oat, and ethanol sensitivity of rice may be lower than that of oat.     

Evidence for bound phytochrome in oat seedlings

Phytochrome is consistently observed in pellets centrifuged from homogenates of etiolated, 5-day-old oat seedlings. The majority of pigment associated with the pellet cannot be removed by buffer washes, nor can appreciable quantities of additional phytochrome be adsorbed onto the sedimented material. Over 70% of phytochrome in the pellet is released by 1% Triton X-100.     

Reciprocity in the activation of geotropism in oat coleoptiles grown on clinostats

Summary The activation of geotropism in oat coleoptiles, grown on horizonta clinostats, i.e., without the tropic influence of gravity, shows a reciprocal relationship between force and time. Two methods were used to approximate geotropic presentation time. In one, this parameter was estimated by extrapolation to zero response from the linear relationship, response = a+b log stimulation time. In the other, stimulation times of very short as well as longer durations were used; under these conditions, the response curve shows two distinct rates, with the lower rate for stimuli of brief duration. The intersection of the two rate-segments of the response curve was taken as the presentation time. Both methods show reciprocity for the activation of geotropism, but yield significantly different reciprocity constants. The ability of the coleoptile to sense gravity is not affected by gravity compensation. With agar as a growth medium, the magnitude of response to gravity is greater than with sand. However, coleoptiles grown in sand are more sensitive to geotropic activation.Work supported by the U. S. Atomic Energy Commission and the National Aeronautics and Space Administration.     

The effects of growth Inhibitors on xylan synthetase activity in oat coleoptiles

Xylan synthetase activity in oat coleoptiles was stimulatedin vivo by pretreatment with the growth inhibitor peroxyacetylnitrate (PAN). However, in vitro treatment of the particulatepreparation with PAN caused enzyme inhibition. Other sulfhydrylreagents, which inhibited growth, such as oxidized glutathioneand parachloromercuribenzoate, also inhibited xylan synthetaseactivity both in vivo and in vitro. It was concluded that thesynthetase is protected internally from PAN and that other enzymeson the pathway to xylan formation may be inhibited by PAN. ¹Present address: Division of Fruit and Vegetable Storage, TheAgricultural Research Organization, The Volcani Center, P.O.B.6, Bet Dagan, Israel. (Received December 5, 1972; )     

Gravity sensing in oat coleoptiles: scatter in growth orientation under different g-conditions

Dark-grown oat seedlings depart from the expected vertical orientation, suggesting that the coleoptile is less responsive to the lateral component of a gravitational stimulus than would be expected. This phenomenon was studied by investigating the gravitropic curvatures of oat (Avena sativa L. cv. Seger) coleoptiles at 10g and over a range of longitudinally applied centripetal accelerations up to 19·4g. In most experiments, the plants were grown and observed at a particular g-level throughout the experiment. Time-lapse video recordings permitted studies of the scatter, measured as the variability of the plants' angle from the vertical (or root mean square value, RMS). The coleoptiles' heights at the end of the experiments were not significantly altered under the centrifugation. Scatter increased with plant age and decreased with increasing g. It decreased in an almost linear fashion as a function of the logarithm of the g-acceleration. In a series of experiments, the g-level was changed from 10g to a higher test g-acceleration. The scatter was then reduced within half an hour after the g-transition. It is pointed out that the experiments confirm that the scatter is g-related but that it was not predicted quantitatively by present theories of the oat coleoptile's gravitropic response kinematics.     

Intracellular localisation of phytochrome and ubiquitin in red-light-irradiated oat coleoptiles by electron microscopy

The intracellular localisation of phytochrome and ubiquitin in irradiated oat coleoptiles was analysed by electron microscopy. We applied indirect immunolabeling with polyclonal antibodies against phytochrome from etiolated oat seedlings or polyclonal antibodies against ubiquitin from rabbit reticulocytes, together with a goldcoupled second antibody, on serial ultrathin sections of resin-embedded material. Immediately after a 5-min pulse of red light-converting phytochrome from the red-absorbing (Pr) to the far-redabsorbing (Pfr) form-the label for phytochrome was found to be sequestered in electron-dense areas. For up to 2 h after irradiation, the size of these areas increased with increasing dark periods. The ubiquitin label was found in the same electrondense areas only after a dark period of 30 min. A 5 min pulse of far-red light, which reverts Pfr to Pr, given immediately after the red light did not cause the electron-dense structures to disappear; moreover, they contained the phytochrome label immediately after the far-red pulse. In contrast, after the reverting far-red light pulse, ubiquitin could only be visualised in the electron-dense areas after prolonged dark periods (i.e. 60 min). The relevance of these data to light-induced phytochrome pelletability and to the destruction of both Pr and Pfr is discussed.Abbreviations FR far-red light; Pfr - Pr far-red-absorbing and red-absorbing forms of phytochrome, respectively - R red light     

Further studies on the role of cell-wall-degrading enzymes in cell-wall loosening in oat coleoptiles

A fungal endo-ß-l,3-glucanase was compared with afungal exo-ß-1,3-glucanase with respect to their effectson elongation and cell-wall extensibility in oat coleoptilesegments. The exo-enzyme enhanced elongation and extensibilityof the cell wall. Its effect was not additive to the effectof indole-3-acetic acid when given together with the latter,at least during 3 hr of incubation. Endo-glucanase showed nosignificant effect on elongation and no interaction with theexo-enzyme. Auxin and exo-glucanase increased extensibilityof the cell wall. The exo-glucanase was separated by isoelectricfocusing. The two fractions which were separated and showedglucanase activity induced elongation and cell wall loosening. (Received March 16, 1970; )     

Kinetics of mitotic rhythmic activity in oat (Avena sativa) coleoptiles following irradiation

The analysis of different intensity (10-200 Gy) irradiation influence on rhythmical processes in mitotic activity of oats coleoptiles was carries out. Obtained results indicate that irradiation disorders phase and amplitude of the cell division rhythm in investigated material. This disturbance, as judged by doze dependence, is associated with unknown molecular structures.     

Separation of tonoplast and plasma membrane potential and resistance in cells of oat coleoptiles

Summary Membrane potential and resistance were recorded from parenchymal cells of oat (Avena) coleoptiles, using one and two intracellular electrodes. Membrane potential is largest (–100 mV) in impalements with low input resistance (2–4 M), and is less negative (–50 mV) in penetrations with high input resistance (> 20 m). The interpretation is that the electrode lodges in the vacuole which is positive to the cytoplasm (but still negative to the external solution), and that measurements of net membrane potential are compromised to varying degrees by leakage shunts introduced across the high resistance vacuolar membrane by the electrode. This conclusion is supported by several additional lines of evidence. (1) It is possible to convert large-R/small-V impalements into small-R/large-V penetrations by passing excess current through the electrode or by briefly ringing the capacitance neutralization circuit in the amplifier. The cells usually recover their resistance in a few minutes, with a concomitant decrease in the negativity of the membrane potential. (2) Changes in external [K] affect the measuree potential by an amount that is independent of the input resistance of the impalement. This is consistent with an effect of [K] _o on the potential of the plasma membrane and the occurrence of leakage shunts primarily at the tonoplast. (3) Quantitatively, the effects of a change in [K] _o on resistance indicate that nearly 90 percent of the input resistance of unshunted cells resides in the tonoplast. (4) The effects of metabolic inhibitors (DNP, CN^–) on potential are smaller in large-R than in small-R impalements. This observation suggests there are electrogenic pumps contributing to the membrane potential at both the plasmalemma and tonoplast. Finally, we conclude that with an electrode in the vacuole it is possible to record potentials that are dominated by the contribution of the plasma membrane, provided care is taken to select impalements combining both large, negative potential and low input resistance.