Oat Coleoptiles Research Articles

Gravitropically‐stimulated seedlings show autotropism in weightlessness

In a spaceflight experiment, autotropism by oat (Avena sativa L.) coleoptiles following gravitropic responses was prominent in weightlessness: counter‐reactions led to the straightening of the curved coleoptiles. This was not the case during clinorotation on earth. The autotropic reactions appeared to be related to the stimulus received during the stimulus period, i.e. the greater the response the greater the autotropic counter‐reaction. Previous models of the gravitropic system which predicted that coleoptiles would not straighten in weightlessness are disproved. A modification to one of the models is proposed which includes the autotropic response observed in spaceflight. The nature of the counter‐reactions in the absence of gravitropic stimulation is discussed.

Role of Expansin in Cell Enlargement of Oat Coleoptiles (Analysis of Developmental Gradients and Photocontrol).

Expansins are wall proteins that mediate a type of acid-induced extension in isolated plant cell walls (S. McQueen-Mason, D.M. Durachko, D.J. Cosgrove [1992] Plant Cell 4: 1425-1433). To assess the role of these proteins in the process of cell enlargement in living tissues, we compared the spatial and temporal growth patterns of oat (Avena sativa L.) coleoptiles with four wall properties related to expansin action. These properties were (a) the ability of isolated walls and living segments to extend in acidic buffer, (b) the ability of heat-inactivated walls to extend upon application of expansins, (c) the amount of immunologically detectable expansin in wall protein extracts, and (d) the extractable expansin activity of walls. Growth rate was maximal in the apical half of dark-grown coleoptiles and negligible in the basal region. This growth pattern correlated with properties a and b; in contrast, the amount and activity of extractable expansin (properties c and d) were reduced only in the most basal region. Upon exposure to white light, coleoptiles abruptly ceased elongation at 8 to 10 h after start of irradiation, and this cessation correlated with reductions in properties a to c. The growth cessation at 8 to 10 h also coincided with the loss of growth response to exogenous auxin and fusicoccin in excised coleoptile segments. These results lend correlative support to the hypothesis that expansin action is important for growth responses of living oat coleoptiles (e.g. responses to acidic buffers, auxin, fusicoccin, aging, and light). Our results suggest that changes in the susceptibility of the wall to expansin action, rather than changes in expansin activity, may be a key determinant of the growth patterns in oat coleoptiles.

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

ABSTRACTDark‐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.

An oat coleoptile wall protein that induces wall extension in vitro and that is antigenically related to a similar protein from cucumber hypocotyls

Plant cell walls expand considerably during cell enlargement, but the biochemical reactions leading to wall expansion are unknown. McQueen-Mason et al. (1992, Plant Cell 4, 1425) recently identified two proteins from cucumber (Cucumis sativus L.) that induced extension in walls isolated from dicotyledons, but were relatively ineffective on grass coleoptile walls. Here we report the identification and partial characterization of an oat (Avena sativa L.) coleoptile wall protein with similar properties. The oat protein has an apparent molecular mass of 29 kDa as revealed by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis. Activity was optimal between pH 4.5 and 5.0, which makes it a suitable candidate for “acid growth” responses of plant cell walls. The oat protein induced extension in walls from oat coleoptiles, cucumber hypocotyls and pea (Pisum sativum L.) epicotyls and was specifically recognized by an antibody raised against the 29-kDa wall-extension-inducing protein from cucumber hypocotyls. Contrary to the situation in cucumber walls, the acid-extension response in heat-inactivated oat walls was only partially restored by oat or cucumber wall-extension proteins. Our results show that an antigenically conserved protein in the walls of cucumber and oat seedlings is able to mediate a form of acid-induced wall extension. This implies that dicotyledons and grasses share a common biochemical mechanism for at least part of acid-induced wall extensions, despite the significant differences in wall composition between these two classes of plants.

Proton efflux from oat coleoptile cells and exchange with wall calcium after IAA or fusicoccin treatment

Elongation growth of plant cells occurs by stretching of cell walls under turgor pressure when intermolecular bonds in the walls are temporarily loosened. The acid-growth theory predicts that wall loosening is the result of wall acidification because treatments (including IAA and fusicoccin) that cause lowered wall pH cause elongation. However, conclusive evidence that IAA primarily reduces wall pH has been lacking. Calcium has been reported to stiffen the cell walls. We have used a microelectrode ion-flux measuring technique to observe directly, and non-invasively, the net fluxes of protons and calcium from split coleoptiles of oats (Avena sativa L.) in unbuffered solution. Normal net fluxes are 10 nmol · m(-2) · s(-1) proton efflux and zero calcium flux. The toxin fusicoccin (1 μM) causes immediate efflux from tissue not only of protons, but also of calcium, about 110 nmol · m(-2) · s(-1) in each case. The data fit the "weak acid Donnan Manning" model for ion exchange in the cell wall. Thus we associate the known "acid-growth" effect of fusicoccin with the displacement of calcium from the wall by exchange for protons extruded from the cytoplasm. Application of 10 μM IAA causes proton efflux to increase transiently by about 15 nmol · m(-2) · s(-1) with a lag of about 10 min. The calcium influx decreases immediately to an efflux of about 20 nmol · m(-2) · s(-1). It appears that auxin too causes an "acid-growth" effect, with extruded protons exchanging for calcium in the cell walls.

Actin and the elongation of plant cells

We have investigated in parallel the effects of different types of inhibitors on elongation of oat coleoptile cells in IAA and on the integrity of the longitudinally oriented actin-containing microfilaments present in control cells as detected by rhodamine phalloidin (RP) staining. Where growth was 50% inhibited by cytochalasin D (CD), we observed extensive to complete breakdown of the microfilaments (MFs) with the appearance of new RP staining in a few nuclei and markedly along the cross walls. When the CD-treated coleoptiles were held at 4°C the nuclei were uniformly strongly stained and cross wall staining was not seen, suggesting that translocation to the nuclei may be an intermediate step in final disposition of the actin. The divalent ions calcium and magnesium both inhibited growth in a dose dependent way, with calcium giving 50% inhibition at 65 mM and magnesium at 25 mM. KCl was not inhibitory and did not reverse the inhibition by divalent ions even at 250 mM. At 50% inhibition by either ion, the long MFs in many cells were replaced either by short fragmented MFs and small brightly staining granules (calcium) or by short usually twisted MFs and large, less intensely staining masses (magnesium). Iodoacetate at 2mM inhibited growth almost completely and resulted in short, fragmented, twisted or curled MFs in most of the cells. Abscisic acid also caused replacement of some MFs with faintly fluorescent bodies somewhat larger than those in CaCl2; occasionally granules similar to those in CaCl2 were also seen. Only mannitol and galactose, which inhibit growth by their osmotic effect, did not cause breakup of the MFs; indeed the MFs in mannitol appeared if anything wider and thicker. The results show that under the influence of three types of growth inhibitors the actin-containing MFs in the cells are broken down to different extents resulting in new structures. The results support the idea that the integrity of the MF bundles is linked, perhaps causally, to the elongation of theAvena cells.

Auxin-induced growth of Avena coleoptiles involves two mechanisms with different pH optima.

Although rapid auxin-induced growth of coleoptile sections can persist for at least 18 hours, acid-induced growth lasts for a much shorter period of time. Three theories have been proposed to explain this difference in persistence. To distinguish between these theories, the pH dependence for auxin-induced growth of oat (Avena sativa L.) coleoptiles has been determined early and late in the elongation process. Coleoptile sections from which the outer epidermis was removed to facilitate buffer entry were incubated, with or without 10 micromolar indoleacetic acid, in 20 millimolar buffers at pH 4.5 to 7.0 to maintain a fixed wall pH. During the first 1 to 2 hours after addition of auxin, elongation occurs by acid-induced extension (i.e. the pH optimum is <5 and the elongation varies inversely with the solution pH). Auxin causes no additional elongation because the buffers prevent further changes in wall pH. After 60 to 90 minutes, a second mechanism of auxin-induced growth, whose pH optimum is 5.5 to 6.0, predominates. It is proposed that rapid growth responses to changes in auxin concentration are mediated by auxin-induced changes in wall pH, whereas the prolonged, steady-state growth rate is controlled by a second, auxin-mediated process whose pH optimum is less acidic.

Comparisons of various biological activities of stereoisomers of methyl jasmonate

Stereoisomers of methyl jasmonate (JA-Me) showed different biological activities in four bioassay systems. Growth of soybean callus was inhibited strongly by (1 R,2 S)- and (1 R,2 R)-JA-Me. By contrast, (1 S,2 R)- and (1 S,2 S)-JA-Me had very low inhibitory effect on it, suggesting that the activity is dependent largely on the (1 R)-configuration. With regard to potato tuber-induction and the senescence-promotion of oat leaves, although the highest activities were found in (1 R,2 S)-JA-Me, isomers which have the (1 S)-configuration showed considerable activities. (1 R,2 S)- and (1 S,2 R)-JA-Me equally inhibited straight growth of oat coleoptiles. These results suggest that requirements of the absolute configurations of the two side chains with respect to the plane of the cyclopentanone ring for each activity are different, and that there are different receptors which trigger reactions leading to the individual activity.

Synergism of action between indolacetic acid (IAA) and highly diluted solutions of CACO 3 on the growth of oat coleoptiles

Synergism of action between indolacetic acid (IAA) and highly diluted solutions of CACO 3 on the growth of oat coleoptiles

Cytochemical Studies on Phytochrome-Mediated Changes of Ca2+Localization in Etiolated Oat Coleoptile Cells

Cytochemical Studies on Phytochrome-Mediated Changes of Ca²⁺Localization in Etiolated Oat Coleoptile Cells

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

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

The outer epidermis of Avena and maize coleoptiles is not a unique target for auxin in elongation growth

A controversy exists as to whether or not the outer epidermis in coleoptiles is a unique target for auxin in elongation growth. The following evidence indicates that the outer epidermis is not the only auxin-responsive cell layer in either Avena sativa L. or Zea mays L. coleoptiles. Coleoptile sections from which the epidermis has been removed by peeling elongate in response to auxin. The magnitude of the response is similar to that of intact sections provided the incubation solution contains both auxin and sucrose. The amount of elongation is independent of the amount of epidermis removed. Sections of oat coleoptiles from which the epidermis has been removed from one side are nearly straight after 22 h in auxin and sucrose, despite extensive growth of the sections. These data indicate that the outer epidermis is not a unique target for auxin in elongation growth, at least in Avena and maize coleoptiles.

Xyloglucan Antibodies Inhibit Auxin-Induced Elongation and Cell Wall Loosening of Azuki Bean Epicotyls but Not of Oat Coleoptiles

Polyclonal antibodies were raised in rabbits against isoprimeverose (Xyl(1)Glc(1)), xyloglucan heptasaccharides (Xyl(3)Glc(4)), and octasaccharides (Gal(1)Xyl(3)Glc(4)). Antibodies specific for hepta- and octasaccharides suppressed auxin-induced elongation of epicotyl segments of azuki bean (Vigna angularis Ohwi and Ohashi cv Takara). These antibodies also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time and the relaxation rate of the cell walls) of azuki segments. However, none of the antibodies influenced auxin-induced elongation or cell wall loosening of coleoptile segments of oat (Avena sativa L. cv Victory). Auxin caused a decrease in molecular mass of xyloglucans in the cell walls of azuki epicotyls and oat coleoptiles. The antibodies inhibited such a change in molecular mass of xyloglucans in both species. Preimmune serum exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosening, or breakdown of xyloglucans. The results support the view that the breakdown of xyloglucans is associated with the cell wall loosening responsible for auxin-induced elongation in dicotyledons. The view does not appear to be applicable to poaceae, because the inhibition of xyloglucan breakdown by the antibodies did not influence auxin-induced elongation or cell wall loosening of oat coleoptiles.

Synthesis, biological activity and metabolism of N-hydroxytryptophan

N-Hydroxytryptophan (α-hydroxyamino-β-indolyl-3-propionic acid) was chemically synthesized. On a mole basis, its relative biological activity in comparison with indole-3-acetic acid at sub-optimal concentrations is: oat coleoptile curvature test=0.129; oat first internode test=0.089 and linseed root growth inhibition test=0.022. Infiltration of N-hydroxytryptophan into oat coleoptile sections leads to the production of indoleacetaldehyde oxime, indoleacetaldehyde and indoleacetic acid. It is suggested that tryptophan could be metabolized in oat coleoptiles to indole-3-acetic acid via N-hydroxytryptophan, indoleacetaldehyde oxime and indoleacetaldehyde.

Partial purification of sequestered particles of phytochrome from oat (Avenu sativa L.) seedlings

Sequestered particles of phytochrome (SAPs) were partially purified from red-light-irradiated oat coleoptiles. Phytochrome pelletability was enhanced by using buffers containing 10 mM Mg(2+) or high concentrations (0.6-0.8 M) of orthophosphate (Pi). Combining the pelletability of phytochrome in the presence of Mg(2+) with that in the presence of 0.6 Pi resulted in a strong enrichment (about 100-fold) of pelletable phytochrome. Antisera were raised against Mg(2+)-Pi-pellets from darkgrown seedlings. Using these antisera, no evidence was found by Western blotting and immunocytochemistry that SAPs contain major proteins other than phytochrome. The major contamination of these enriched SAP preparations consisted of protein crystals which are probably catalase. The preparations contained methyltransferase and protein-kinase activities which were not associated with SAPs. Phytochrome purified from SAPs served as a substrate for protein-kinase activity but not for the methyltransferase activity. Phytochrome itself did not show any kinase activity.

Phototropism: mechanisms and ecological implications

Abstract. Phototropism in seed plants, either etiolated or de‐etiolated, is mediated by unidentified photoreceptor(s) sensitive to blue and near‐UV regions of the light spectrum. Green plants may have an additional phototropic system sensitive to red light. Fluence‐response studies of the blue light‐sensitive phototropism, initially made on oat coleoptiles, have indicated the occurrence of multiple response types. Of those, two are found to be general: the first pulse‐induced positive phototropism (fPIPP), or the so‐called first positive curvature, and the time‐dependent phototropism (TDP) or the second positive curvature. The fPIPP, elicited by a pulse stimulus shorter than a few minutes, is characterized by a bell‐shaped fluence‐response curve and the validity of reciprocity. The TDP, elicited by prolonged irradiation, is characterized by its dependence on the exposure time and the invalidity of reciprocity. Studies made on these two response types have revealed the following: (1) plants acquire directional light information for phototropism by sensing internal light gradients created by light scattering and absorption; (2) phototropism results from redistribution of growth, i.e. inhibition on the irradiated side and compensating stimulation on the shaded side; (3) lateral movement of growth regulators, the principle of the Cholodny‐Went theory, can account for the growth redistribution, and auxin is clearly the mediating regulator in maize coleoptiles. This review further describes some mechanistic implications of fPIPP. Experimental results indicate that (1) fPIPP is mediated by a single step of photoreaction, (2) the responsiveness, reflected in the height of the fluenceresponse curve, is reduced by pre‐irradiation with blue light and recovers gradually afterward, and (3) the light sensitivity, reflected in the position of the fluence‐response curve along the log fluence axis, is also reduced by the pre‐irradiation and recovers gradually. Analyses of these results, based on kinetic models, suggest that the bell‐shaped fluence‐response curve is caused by the difference in the amounts of a photoproduct between irradiated and shaded sides, and that fPIPP represents a mechanism of TDP. It is also indicated that phytochrome in the red‐absorbing form exerts two separate effects on phototropism: reduction of the light sensitivity and enhancement of the responsiveness. Along with the discussion of the mechanisms of phototropism, their ecological implications are considered.

Intracellular localisation of phytochrome in oat coleoptiles by electron microscopy

Intracellular localisation of phytochrome in oat coleoptiles by electron microscopy

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-grown Avena 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.

The influence of the herbicide mefenacet on cell division and cell enlargement in plants

Summary:The herbicide mefenacet has recently been developed for pre–emergence control of graminaceous and some dicotyledonous weeds in rice. Whereas mefenacet is known to inhibit root growth, its effect on the components of growth, cell division and cell enlargement, have not been determined. The purpose of this study was to evaluate the influence of mefenacet on growth, cell enlargement and cell division in Chlamydomonas (a green alga) and oats. Cell division in Chlamydomonas was completely inhibited and cell enlargement was inhibited by an average of 18% during the first cell cycle period when cells were treated with 6–7 μM mefenacet. The rate of oat root growth in sand culture between 12 and 24 h after initiation of treatment was reduced 90% at 10 μM mefenacet. Cell enlargement of oat coleoptile sections in culture solution was inhibited 25% at 10 μM. Mefenacet inhibited mitotic entry rather than disrupted the mitotic sequence. Cell division in oat roots was inhibited 54% by 10 μM mefenacet. This shows cell division inhibition occurs in G1, S, or G2 of the cell cycle. The action of mefenacet is most like that of the chloroacetamides among the herbicide classes known to inhibit growth.

In vitro selection for 2,4-D tolerance in red clover

In vitro, selection is a viable method of selecting herbicide-tolerant crops. This research was to evaluate in vitro selection techniques for enhancing 2,4-D [(2,4-dichlorophenoxy) acetic acid] tolerance in red clover (Trifolium pratense L.). In vivo and in vitro responses to 2,4-D of eight diverse red clover populations were correlated (r=0.77), justifying in vitro selection for 2,4-D tolerance. Suspension cultures of a red clover genotype capable of regeneration were plated onto agar-based nutrient media supplemented with 0.18 mM 2,4-D for selection experiments. After two cycles of selection, 16 2,4-D tolerant callus lines were identified based on visual growth assessment. These lines were evaluated for 2,4-D tolerance (based on 2,4-D content), using a 2,4-D bioassay procedure which consisted of placing selected callus tissue pieces on top of oat (Avena sativa L.) coleoptile or internode sections. The relative amount of 2,4-D in the callus tissue was estimated by the amount of oat section elongation after 24 h. Two of the more tolerant callus lines had 61% and 83% less 2,4-D in their tissues than the susceptible control tissue. These studies indicated that in vitro selection can enhance the levels of 2,4-D tolerance in red clover callus tissue.