Isolation and identification of a light-induced growth inhibitor in diffusates from blue light-illuminated oat (Avena sativa L.) coleoptile tips

The amounts of two growth inhibitors in diffusates from illuminatedhalves of phototropically stimulated oat (Avena sativa L.)coleoptile tips were larger than those from shaded halves. The less polarinhibitor was isolated from diffusates from oat coleoptile tips illuminatedwithblue light, and identified as uridine from ¹H NMR spectrum. Thedistribution of endogenous uridine in diffusates from the illuminated andshadedsides of coleoptile tips unilaterally exposed to blue light for 3, causing a first positive phototropic curvature, and fromdark-control tips, was determined using a physicochemical assay. The uridineconcentration was significantly higher in the diffusates from the illuminatedside than in those from the shaded side and the dark-control. Uridine inhibitedthe growth of etiolated oat coleoptile tips at concentrations of 30 and above. These results suggest that uridine plays a role inthe phototropism of oat coleoptiles.     

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.     

Mesocotyl growth of etiolated seedlings ofAvena sativa andZea mays in relation to light and diffusible auxin

Diffusible auxin levels were measured in coleoptiles and mesocotyls of dark-grown seedlings ofavena sativa (cv. Spear) andZea mays (cv. Golden Cross Bantam) using theAvena curvature bioassay. The coleoptile tip was confirmed as the major auxin source in etiolated seedlings. Auxin levels were found to decrease basipetally in sequent sections of theAvena coleoptile but not to decrease in apical sections of increasing length. An inhibitor capable of inducing positive curvatures ofAvena test coleoptiles was discovered in diffusates from the mesocotyls of oat and corn seedlings. The amount of this inhibitor was correlated with the cessation of mesocotyl growth of oat seedlings grown in darkness, and with the inhibition of mesocotyl growth of corn seedlings exposed to red light.     

Response of actin microfilaments during phytochrome-controlled growth of maize seedlings

Summary In seedlings of maize (Zea mays L. cv. Percival), growth is controlled by the plant photoreceptor phytochrome. Whereas coleoptile growth is promoted by continuous far-red light, a dramatic block of mesocotyl elongation is observed. The response of the coleoptile is based entirely upon light-induced stimulation of cell elongation, whereas the response of the mesocotyl involves light-induced inhibition of cell elongation. The light response of actin microfilaments was followed over time in the epidermis by staining with fluorescence-labelled phalloidin. In contrast to the underlying tissue, epidermal cells are characterized by dense longitudinal bundles of microfilaments. These bundles become loosened during phases of rapid elongation (between 2–3 days in irradiated coleoptiles, between 5–6 days in dark-grown coleoptiles). The condensed bundles re-form when growth gradually ceases. The response of actin to light is fast. If etiolated mesocotyls are transferred to far-red light, condensation of microfilaments can be clearly seen 1 h after the onset of stimulation together with an almost complete block of mesocotyl elongation. The observations are discussed in relation to a possible role of actin microfilaments in the signal-dependent control of cell elongation.     

The Rice COLEOPTILE PHOTOTROPISM1 gene encoding an ortholog of Arabidopsis NPH3 is required for phototropism of coleoptiles and lateral translocation of auxin

We isolated a mutant, named coleoptile phototropism1 (cpt1), from gamma-ray-mutagenized japonica-type rice (Oryza sativa). This mutant showed no coleoptile phototropism and severely reduced root phototropism after continuous stimulation. A map-based cloning strategy and transgenic complementation test were applied to demonstrate that a NPH3-like gene deleted in the mutant corresponds to CPT1. Phylogenetic analysis of putative CPT1 homologs of rice and related proteins indicated that CPT1 has an orthologous relationship with Arabidopsis thaliana NPH3. These results, along with those for Arabidopsis, demonstrate that NPH3/CPT1 is a key signal transduction component of higher plant phototropism. In an extended study with the cpt1 mutant, it was found that phototropic differential growth is accompanied by a CPT1-independent inhibition of net growth. Kinetic investigation further indicated that a small phototropism occurs in cpt1 coleoptiles. This response, induced only transiently, was thought to be caused by the CPT1-independent growth inhibition. The 3H-indole-3-acetic acid applied to the coleoptile tip was asymmetrically distributed between the two sides of phototropically responding coleoptiles. However, no asymmetry was induced in cpt1 coleoptiles, indicating that lateral translocation of auxin occurs downstream of CPT1. It is concluded that the CPT1-dependent major phototropism of coleoptiles is achieved by lateral auxin translocation and subsequent growth redistribution.     

Inhibitory action of red light on the growth of the maize mesocotyl: evaluation of the auxin hypothesis

Brief irradiation of 3-d-old maize (Zea mays L.) seedlings with red light (R; 180 J m^-2) inhibits elongation of the mesocotyl (70–80% inhibition in 8 h) and reduces its indole-3-acetic acid (IAA) content. The reduction in IAA content, apparent within a few hours, is the result of a reduction in the supply of IAA from the coleoptile unit (which includes the shoot apex and primary leaves). The fluence-response relationship for the inhibition of mesocotyl growth by R and far-red light closely resemble those for the reduction of the IAA supply from the coleoptile. The relationship between the concentration of IAA (1–10 M) supplied to the cut surface of the mesocotyl of seedlings with their coleoptile removed and the growth increment of the mesocotyl, measured after 4 h, is linear. The hypothesis that R inhibits mesocotyl growth mainly by reducing the IAA supply from the coleoptile is supported. However, mesocotyl growth in seedlings from which the coleoptiles have been removed is also inhibited by R (about 25% inhibition in 8 h). This inhibition is not related to changes in the IAA level, and not relieved by applied IAA. In intact seedlings, this effect may also participate in the inhibition of mesocotyl growth by R. Inhibition of cell division by R, whose mechanism is not known, will also result in reduced mesocotyl elongation especially in the long term (e.g. 24 h).Abbreviations FR far-red light - IAA indole-3-acetic acid - P_fr phytochrome in the far-red-absorbing form - P_r phytochrome in the red-absorbing form - R red light     

Gravitropism and Phototropism of Maize Coleoptiles: Evaluation of the Cholodny-Went Theory Through Effects of Auxin Application and Decapitation

It was investigated whether or not gravitropism and phototropismof maize (Zea mays L.) coleoptiles behave as predicted by theCholodny-Went theory in response to auxin application, decapitationand combinations of these treatments. Gravitropism was inducedat an angle of 30° from the vertical, and phototropism,by a pulse of unilateral blue light. Either tropism of the coleoptilewas inhibited by IAA, applied as a ring of IAA-lanolin pasteto its sub-apical part, and by decapitation. The dose-responsecurves for the effects of applied IAA on tropisms and growthof intact coleoptiles as well as the time courses of tropismsinduced in decapitated coleoptiles could be explained by thethree conclusions in the literature: (1) the tip of the coleoptileis the site of auxin production, (2) lateral translocation ofauxin in gravitropism occurs along the length of the coleoptile,and (3) lateral translocation of auxin in phototropism occursin the coleoptile tip. By examining the effects of decapitationmade at different distances from the top and of IAA appliedto the cut surface of decapitated coleoptiles, it was indicatedthat auxin is produced in the apical 1 mm zone of an intactcoleoptile and that lateral auxin translocation for phototropismtakes place in an apical part that somewhat exceeds the zoneof auxin production. (Received October 14, 1994; Accepted December 26, 1994)     

The Effects of Phytohormones and 5-Azacytidine on Apoptosis in Etiolated Wheat Seedlings

The development of etiolated wheat (Triticum aestivum L.) seedlings is necessarily accompanied by apoptosis in their coleoptiles and first leaves. Internucleosome DNA fragmentation, which is characteristic of apoptosis, was detected in the coleoptile as soon as six days after germination. After eight days of germination, DNA fragmentation was clearly expressed in the coleoptile and was noticeable in the apical part of the first-leaf blade. Growing of intact seedlings or incubation of their shoots in the presence of such phytohormones as benzyladenine, gibberellin A₃, fusicoccin C, and 2,4-D at the concentration of 10^–5 M did not essentially affect DNA fragmentation in the coleoptile. As distinct from antioxidants, none of the phytohormones used prevented apoptosis in wheat seedlings. In contrast, ABA (10^–5 M) and an ethylene producer, ethrel (2-chloroethylphosphonic acid, 10^–2–10^–3 M), stimulated sharply DNA fragmentation in the coleoptile. An inhibitor of DNA methylation, 5-azacytidine, was very efficient in the stimulation of DNA fragmentation in the coleoptiles of eight-day-old seedlings at its concentration of 100 g/ml. Thus, some phytohormones can regulate apoptosis, and DNA methylation is involved in this process. Our results indicate that apoptosis activation by some phytohormones may be mediated by their regulation of DNA methylation/demethylation, which is responsible for the induction of genes encoding apoptogenic proteins and/or the repression of antiapoptotic genes.     

Effects of submergence on development and gravitropism in the coleoptile of Oryza sativa L.

Caryopses of rice (Oryza sativa L. cv. Sasanishiki) were germinated in air or under water. In submerged seedlings a twofold increase in coleoptile growth rate and an inhibition of root growth was observed. The amount of starch in the amyloplasts of submerged coleoptiles was substantially reduced compared to the air-grown control plants and plastids had a proplastidic character. During the rapid elongation of coleoptiles under water, the osmotic concentration of the press sap remained constant, whereas in air-grown coleoptiles a decrease was measured. Determination of curvature of gravistimulated air-grown and submerged shoots was carried out by placing the coleoptiles horizontally in air of 98% relative humidity. Air-grown coleoptiles reached a vertical orientation within 5 h after onset of gravistimulation. In coleoptiles germinated under water the first signs of consistent negative gravitropic bending occurred after 4–5 h and curvature was complete after 24 h. During the first 5 h of gravistimulation the water-grown coleoptiles grew at an average rate of 0.39 mm·h^–1, whereas in air-grown coleoptiles a rate of 0.27 mm·h^–1 was measured. Concomitant with the delayed onset of gravitropic bending of the water-grown coleoptiles, a change in plastid ultrastructure and an increase in starch content was observed. We conclude that the gravitropic responsiveness of the rice coleoptile depends on the presence of starch-filled amyloplasts.We wish to thank H.-J. Ensikat for technical assistance with the scanning electron microscopy. Supported by the Bundesminister für Forschung und Technologie and the Deutsche Forschungsgemeinschaft.     

IAA transport during the phototropic responses of intact Zea and Avena coleoptiles

Summary Transport of indolyl-3-acetic acid (IAA) was studied during the phototropic responses of intact shoots and detached coleoptiles of Zea mays L. and Avena sativa L. The use of a high specific activity [5-³H]IAA and glass micropipettes enabled asymmetric application of the IAA to be made to individual coleoptiles with minimal tissue damage.A unilateral stimulus of 2.59×10^-11 einstein cm^-2 of blue light, probably in the dose range of the first positive phototropic response, caused significant net lateral movement of radioactivity from [5-³H]IAA away from the illuminated side of intact shoots and detached coleoptile apices of both Avena and Zea. The magnitude of the net lateral movement was 15.3% in Zea seedlings and 12.3% in Avena seedlings. Chromatographic analyses indicated that the movement of radioactivity reflected that of IAA. A phototropic stimulus of 1.24×10^-7 einstein cm^-2, which was probably in the second positive dose range, caused significant lateral movement of radioactivity in intact shoots and detached coleoptiles of Zea but not of Avena.In intact Zea seedlings, neither phototropic dosage affected the longitudinal transport of IAA. In intact Avena seedlings, first positive stimulation inhibited longitudinal transport only when the IAA was applied to the illuminated side of the coleoptile, but second positive stimulation inhibited basipetal movement of IAA regardless of the side of application.Exposing the intact seedlings to red light before phototropic stimulation abolished lateral transport after a first positive stimulus in Zea and in Avena.Phototropic stimulation can thus induce a lateral transport of IAA towards the shaded side of the coleoptiles of both Zea and Avena seedlings, and can affect longitudinal movement of IAA in the coleoptile of Avena. However, since phototropic curvature was observed under certain conditions in the absence of either of these effects, the extent to which they are involved in the induction of asymmetric growth in a stimulated coleoptile has yet to be resolved.     

The lateral transport of IAA in intact coleoptiles of Avena sativa L. and Zea mays L. during geotropic stimulation

Summary Movement of IAA was studied in excised coleoptile apices and whole seedlings of Zea mays L. and Avena sativa L. during geotropic stimulation. A micropipette technique permitted the application of [5-³H]IAA at predetermined points on the coleoptiles with minimal tissue damage.When [5-³H]IAA was applied to the upper side of a horizontal excised Zea coleoptile, about 60% of the recoverable radioactivity had moved into the lower half after 2 h. In contrast, when application was made to the lower side of a horizontal excised coleoptile, only 4% of the radioactivity migrated to the upper half. There was, thus, a net downward movement of 56%. Similar patterns of distribution were found for radioactivity in both the tissue and the basal receiver blocks. In horizontal shoot tissues of intact Zea seedlings a net downward movement of about 30% of the recoverable radioactivity occurred after 1 h of geotropic stimulation. Comparable experiments with Avena indicated a net downward movement of 6–12% in excised apices of coleoptiles and in the intact shoot. In both Zea and Avena chromatographic analyses of tissue and receiver blocks indicated that the movement of radioactivity reflected that of IAA.In Zea coleoptiles, the lateral migration of radioactivity after 2 h was 3 to 4 times greater in the apical tissues than in the basal tissues. A significant net downward movement of radioactivity was detected after 10 min of geotropic stimulation in the extreme apex of Zea coleoptiles but not in the more basal regions.These experiments show that downward lateral transport of IAA occurs in intact shoots of Zea and Avena seedlings upon geotropic stimulation. Lateral transport of IAA had previously been demonstrated only in sub-apical segments of Zea coleoptiles.     

Preferential Induction of Alcohol Dehydrogenase in Coleoptiles of Rice Seedlings Germinated in Submergence Condition

Difference in the growth response to submergence between coleoptiles and roots of rice (Oryza sativa L.) was investigated in 9-d-old rice seedlings. The coleoptile length in the submergence condition was much greater than that in aerobic condition, whereas the root length in the submergence condition was less than that in the aerobic condition. Alcohol dehydrogenase (ADH) activity in the coleoptiles in the submergence condition was much greater than that in the aerobic condition, but ADH activity in the roots in the submergence condition increased slightly. These results suggest that the preferential ADH induction in rice seedlings may contribute to the difference in the growth response between the coleoptiles and roots under low oxygen conditions.     

Mesocotyl growth of etiolated seedlings ofAvena sativa andZea mays in relation to light and diffusible auxin

Diffusible auxin levels were measured in coleoptiles and mesocotyls of dark-grown seedlings ofavena sativa (cv. Spear) andZea mays (cv. Golden Cross Bantam) using theAvena curvature bioassay. The coleoptile tip was confirmed as the major auxin source in etiolated seedlings. Auxin levels were found to decrease basipetally in sequent sections of theAvena coleoptile but not to decrease in apical sections of increasing length. An inhibitor capable of inducing positive curvatures ofAvena test coleoptiles was discovered in diffusates from the mesocotyls of oat and corn seedlings. The amount of this inhibitor was correlated with the cessation of mesocotyl growth of oat seedlings grown in darkness, and with the inhibition of mesocotyl growth of corn seedlings exposed to red light.     

Experimental Modification of Pigment Content and Phototropic Sensitivity in Excised Avena Coleoptiles

Avena seedlings were imbibed and germinated in the presence of inhibitors of carotenoid biosynthesis. After excision and defoliation, the coleoptiles were cultured in the presence of these basally supplied inhibitors and their growth, phototropic behavior and pigment content were subsequently measured. Total carotenoids could be reduced to ca. 20 percent of the control value without marked influence on the dose-response curve for the first positive curvature. Chromatographic analysis of extracted carotenoids on alumina columns revealed that the inhibitors produced both qualitative and quantitative changes, reducing one fraction and virtually eliminating two others. The total riboflavin content of the coleoptiles was almost completely unaffected by these treatments. The data are applied to an analysis of the nature of the photoreceptor in phototropism of the Avena coleoptile.     

Inhibition of the Coleoptile Growth in Avena sativa by Endosperm Removal--Changes in Auxin, Osmotica and Cell Wall

Removal of the endosperm from 84-h-old etiolated oat seedlingsstrongly retarded the subsequent growth of coleoptiles. Thecontribution of the endosperm to coleoptile growth was studied.Endosperm removal was found to: (1) decrease the endogenouslevel of indole-3-acetic acid (IAA) in the coleoptile tip. IAAapplied to the coleoptile tip stimulated coleoptile growth inseedlings with and without the endosperm. The sensitivity ofthe coleoptile to a suboptimal concentration of IAA was higherin seedlings without the endosperm than in intact ones. At theoptimal concentration of IAA, however, the final length of thecoleoptile was larger in intact seedlings than in those withoutthe endosperm. (2) decrease the concentration of the solublesugars and amino acids in the cell sap. (3) retard the increasein the amount of polysaccharides in the cell wall of the coleoptile,particularly noncellulosic ones. (4) make the cell wall mechanicallyrigid according to stress-relaxation analysis of the cell wall.(5) induce an increase in the osmotic potential of the coleoptilecell sap. From these results, it was concluded that the endosperm suppliesthe coleoptile with IAA, sugars and amino acids, thus promotingcoleoptile growth. (Received September 24, 1987; Accepted February 3, 1988)     

Phototropic stimulation does not induce unequal distribution of indole-3-acetic acid in maize coleoptiles

Distribution of endogenous diffusible auxin into agar blocks from phototropically stimulated maize coleoptile tips was studied using a bioassay and a physicochemical assay, to clarify whether phototropism in maize coleoptiles involves a lateral gradient in the amount of auxin. At 50 min after the onset of phototropic stimulation, when the phototropic response was still developing, direct assay of the blocks with the Avena curvature test showed that the auxin activity in the blocks from the shaded half-tips was twice that of the lighted side, at both the first and second positive phototropic curvatures. However, physicochemical determination following purification showed that the amount of indole-3-acetic acid (IAA) was evenly distributed in the blocks from lighted and shaded coleoptile half-tips at both the first and second positive phototropic curvatures. The even distribution of the IAA was also confirmed with the Avena curvature test following purification by HPLC. These results indicate that phototropism in maize coleoptiles is not caused by a lateral gradient of IAA itself and thus cannot be described by the Cholodny-Went theory. Furthermore, the lower auxin activity in the blocks from the lighted half-tips suggests the presence of inhibitor(s) interfering with the action of auxin and their significant diffusion from unilaterally illuminated coleoptile tips.     

Revision of the theory of phototropism in plants: a new interpretation of a classical experiment

Went's classical experiment on the diffusion of auxin activity from unilaterally illuminated oat coleoptile tips (Went 1928), was repeated as precisely as possible. In agreement with Went's data with theAvena curvature assay, the agar blocks from the illuminated side of oat (Avena sativa L. cv. Victory) coleoptile tips had, on an average, 38% of the auxin activity of those from the shaded side. However, determination of the absolute amounts of indole-3-acetic acid (IAA) in the agar blocks, using a physicochemical assay following purification, showed that the IAA was evenly distributed in the blocks from the illuminated and shaded sides. In the blocks from the shaded and dark-control halves the amounts of IAA were 2.5 times higher than the auxin activity measured by theAvena curvature test, and in those from the illuminated half even 7 times higher. Chromatography of the diffusates prior to theAvena curvature test demonstrated that the amounts of two growth inhibitors, especially of the more polar one, were significantly higher in the agar blocks from the illuminated side than in those from the shaded side and the dark control. These results show that the basic experiment from which the Cholodny-Went theory was derived, does not justify this theory. The data rather indicate that phototropism is caused by the light-induced, local accumulation of growth inhibitors against a background of even auxin distribution, the diffusion of auxin being unaffected.Abbreviation IAA indole-3-acetic acid     

Can Lateral Redistribution of Auxin Account for Phototropism of Maize Coleoptiles?

Elongation growth of intact, red-light grown maize (Zea mays L.) coleoptiles was studied by applying a small spot of an indole acetic acid (IAA)-lanolin mixture to the coleoptile tip. We report that: (a) endogenous auxin is limiting for growth, (b) an approximately linear relation holds between auxin concentration and growth rate over a range which spans those rates occurring in phototropism, and (c) an auxin gradient established at the coleoptile tip is well sustained during its basipetal transport. We argue that the growth differential underlying coleoptile phototropism (first-positive curvature) can be explained by redistribution of auxin at the coleoptile tip.     

Levels of indole-3-acetic acid in intact and decapitated coleoptiles as determined by a specific and highly sensitive solid-phase enzyme immunoassay

A specific solid-phase enzyme immunoassay for the detection of as little as 3–4 pg of indole-3-acetic acid (IAA) is described. The assay involves minimal procedural efforts and requires only standard laboratory equipment. Up to 50 samples in triplicate, processed simultaneously, can be assayed and evaluated in 2.5 h. As little as 1 mg oat coleoptile tissue is sufficient for a quantitative IAA analysis and little or no extract purification is necessary. Using this assay, levels of IAA have been determined in coleoptiles of maize and oat. The distribution of IAA within single coleoptiles was quantitated and the production of IAA during the regeneration of the physiological tip in Avena coleoptiles was investigated. The changes in levels of IAA and other major phytohormones were quantitated during the growth of oat coleoptiles.Abbreviations ABA abscisic acid - BHT butylated hydroxytoluene - BSA bovine serum albumin - IAA indole-3-acetic acid - TBS Trishydroxymethylaminomethane buffered saline Part 21 in the series Use of Immunoassay in Plant Science     

Effects of ethanol on growth of rice seedlings

Effects of ethanol, the end product of ethanolic fermentation, on the growth of rice (Oryza sativa L.) seedlings were determined as a means of evaluating growth responses under anoxia. The ethanol concentrations in roots and coleoptiles of the seedlings subjected to 48 h-anoxia, and in their culture medium were 23 and 32 µmol g^–1 fresh weight, and 19 µmol ml^–1, respectively. The growth of the roots and coleoptiles of the seedlings was restricted by exogenous ethanol at concentrations above 50 mM and 100 mM, respectively, suggesting that the roots are more sensitive to ethanol than the coleoptiles.