Metabolism of Indole-3-acetaldehyde

Infiltration of indolcacelaldehyde (IAAId) into living tissues of sonic lower and higher plants gives rise simultaneously to both indoleacetic acid (IAA) and Iryptophol (T-ol). But on a molar basis, there is no correlation between the products indicating a disimitation. Expressed juice of Avena coleoptiles by itself, exhibits only IAA forming activity. Approximately two moles of IAAld are consumed for each mole of IAA formed at pH 4.5, but only if necessary corrections are made for losses of substrate and products. Addition of reduced NAD or NADP readily induces tryptophol formation. But even at pH 4.5, adding reduced NADP causes greater tryptophol formation, leading to a marked divergence in the acid-alcohol ratio. Varying the pH in the presence of reduced coenzymes also alters the ratio, with alcohol formation predominating. NAD and NADP have no influence on the formation of IAA from IAAld by the whole cytoplasm of Avena coleoptiles. Whole cytoplasm of Asparagus shoots forms both IAA and tryptophol from IAAld, but in widely varying amounts, devoid of any suggestive stoichiometry between the products. With the acetone powder of Avena coleoptiles including the first leaf, data indicating an apparent disimitation of IAAld are obtainable only at pH 4.5. On altering the pH however, unequal amounts of the two products, namely IAA and tryptophol, are formed and hence a different ratio results. Acetone powders of wheat coleoptiles and Asparagus shoots do not yield data supporting disimitation either at pH 4.5 or 7.2. IAA formation in Avena is aerobic while tryptophol formation is seemingly independent of oxygen supply. The former activity is selectively abolished by 10^?3M dithionite while the latter activity suffers a similar suppression in the presence of 10^?3M manganese sulphate. Varying the IAAld concentration results in unequal amounts of the two products, revealing the dissimilar affinity of the two activities for the common substrate Saturation to a level of 30 percent with ammonium sulphate throws out most of the acid-forming activity whereas the alcohol-forming system appears mostly in the protein fraction precipitated between 30 and 40 percent saturation. The enzyme system of Avena coleoptiles oxidizing IAAld to IAA can also be easily separated by Sephadex gel filtration and its independent activity demonstrated in the total absence of tryptophol formation. Based on the heterogeneous properties of the two activities, metabolism of IAAld in Avena coleoptiles is believed to be mediated by two independent enzyme systems without the intervention of a mutase or a dismutation mechanism.     

Effects of calmodulin antagonists on auxin-stimulated proton extrusion in Avena sativa coleoptile segments

The effect of the 5 calmodulin (CaM) antagonists trifluoperazine (TFP). compound 48/80, N-(6-aminohexyl)-naphthalenesulfonamtde (W-5), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and calmidazolium on auxin-dependent medium acidification was investigated in abraded segments of Avena sativa L. cv. Victory I. Buffering capacity, Asn content, and changes in pH of bathing solutions were measured in the presence of these inhibitors. When coleoptiles were treated with TFP or compound 48/80, the Asn content and the buffering capacity increased, thus suggesting that plasma membrane permeability was modified. On the contrary. the effect of calmidazolium, W-5. and W-7 on Asn release and buffering capacity was rather low; only small effects being observable at the highest concentration employed. Calmidazolium and W-7 strongly inhibited auxin-dependent medium acidification. W-5 did not affect medium acidification. The specificity of these CaM antagonists and their effects on medium acidification are discussed. The data adduced is consistent with the working hypothesis which postulates an essential role for the Ca²⁺-CaM system on auxin-dependent medium acidification.     

Antibodies as probes for the study of location and metabolism of (1→3), (1→4)-β-D-glucans

Polyclonal antibodies, raised against ((1→3), (1→4)-β-D-glucans from oat ( Avena sativa L.) caryopsis, were used to investigate the location and the metabolism of mixed-linked β-D-glucans. The binding of these antibodies to the cell walls of oat coleoptiles was shown by an indirect fluorescence method. Distinct fluorescent regions were observed along the inner layers of the walls of each cell. The preimmune serum or antibodies pretreated with oat caryopsis β-D-glucans did not react with the cell walls. Glucan antibodies were bound to the walls of other Poaceae coleoptiles as well as to those from oat mesocotyls and roots, whereas they were not bound to the walls of some dicotyledons tested. The relative glucan content of the cell walls of oat coleoptiles as determined by β-D-glucanase (EC 3.2.1.73) treatment was maximum between day 3 and 4 after soaking, but it declined during further elongation. A rapid decrease in glucan content was observed in excised coleoptiles when auxin or β-D-glucanase was present. There was a clear correlation between the glucan content expressed on a basis of cell wall polysaccharides and the amount of the antibodies bound to the cell walls. These results indicate that the antibodies are useful probes to detect and determine (1→3), (1→4)-β-D-glucans of cell walls.     

Effect of cycloheximide on IAA-induced proton excretion and IAA-induced growth in abraded Avena coleoptiles

IAA-induced proton excretion in peeled or abraded oat ( Avena saliva L. cv. Victory) coleoptiles is closely associated with IAA-induced growth. It was attempted to separate these two processes by using cycloheximide to inhibit them differentially. Growth of abraded coleoptile segments was measured by a shadow graphic method, and their IAA-induced acidification of the external solution was monitored with a pH meter. IAA stimulated proton excretion in abraded Avena coleoptile segments after a 13 min lag. IAA-induced proton excretion was inhibited within 5 min by cycloheximide at concentrations of 1.8 × 10⁻⁶, 3.6 × 10 or 3.6 × 10⁻⁵ M. Cycloheximide at these concentrations, added within 4 min of IAA, prevented IAA-induced acidification of the medium for at least 60 min. However, it did not prevent IAA-induced growth during this time. It is concluded that some of the initial IAA-induced growth seen in Avena coleoptiles is independent of detectable IAA-induced proton excretion.     

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.     

Production of auxins by bacteria isolated from the roots of pine seedlings (Pinus silvestris L.).

Qualitative and quantitative studies were carried out on the production of auxins by Coryneform bacteria, the only bacterial types isolated from roots of pine seedlings. Almost all isolates were capable of producing auxins in tryptophan containing media. In media without this amino acid only trace or no auxins were produced. Most of the bacteria studied synthesized auxins located on the chromatograms run with isopropanol, ammonia, water (10:1:1 v/v) at Rf 0.3--0.5. Moreover substances with Rf values 0.05--0.2 and 0.8--1.0 were produced by some strains. No plant growth inhibitors detected with the Avena coleoptiles biotest were produced by the bacteria studied.     

Immunogold electron microscopy of phytochrome in Avena: identification of intracellular sites responsible for phytochrome sequestering and enhanced pelletability

Using monoclonal antibodies to the plant photoreceptor, phytochrome, we have investigated by immunogold electron microscopy the rapid, red light-induced, intracellular redistribution (termed "sequestering") of phytochrome in dark-grown Avena coleoptiles. Pre-embedding immunolabeling of 5-micron-thick cryosections reveals that sequestered phytochrome is associated with numerous, discrete structures of similar morphology. Specific labeling of these structures was also achieved by post-embedding ("on-grid") immunostaining of LR-White-embedded tissue, regardless of whether the tissue had been fixed chemically or by freeze substitution. The phytochrome-associated structures are globular to oval in shape, 200-400 nm in size, and are composed of amorphous, granular material. No morphologically identifiable membranes are present either surrounding or within these structures, which are often present as apparent aggregates that approach several micrometers in size. An immunogold labeling procedure has also been developed to identify the particulate, subcellular component with which phytochrome is associated in vitro as a consequence of irradiation of Avena coleoptiles before their homogenization. Structures with appearance similar to those identified in situ are the only components of the pelletable material that are specifically labeled with gold. We conclude that the association of phytochrome with these structures in Avena represents the underlying molecular event that ultimately is expressed both as red light-induced sequestering in vivo and enhanced pelletability of phytochrome detected in vitro.     

Static electric fields do not cause coleoptile movements

A photodiode array method has been used to record automatically minute lateral movements of Avena sativa L. (cv. Seger I) coleoptiles in a static electric field. The electric fields, 600 or 5000 V cm⁻¹ did not cause any significant movement of the coleoptiles, in contrast to reports in the literature. Likewise, in experiments where photographic recordings were made, no significant movements were found.     

Characterization of protein and transcript levels of the chaperonin containing tailless complex protein-1 and tubulin during light-regulated growth of oat seedlings

In grass seedlings the network of cortical microtubules is reorganized during light-dependent growth of coleoptiles and mesocotyls. We investigated the effects of light-dependent growth on the relative steady-state levels of the mRNAs and protein levels of alpha-tubulin and the epsilon-subunit of the chaperonin containing tailless complex protein-1 in oat (Avena sativa) coleoptiles, which were grown in different light conditions to establish different growth responses. The soluble pools of the epsilon-subunit of the chaperonin containing tailless complex protein-1 and alpha-tubulin decreased in nonelongating coleoptiles, suggesting that the dynamics of the light-regulated soluble pool reflect the processes occurring during reorganization of cortical microtubules. The shifts in pool sizes are discussed in relation to the machinery that controls the dynamic structure of cortical microtubules in plant cells.     

Gravitropic responses of the Avena coleoptile in space and on clinostats. I. Gravitropic response thresholds

We conducted a series of gravitropic experiments on Avena coleoptiles in the weightlessness environment of Spacelab. The purpose was to test the threshold stimulus, reciprocity rule and autotropic reactions to a range of g-force stimulations of different intensities and durations The tests avoided the potentially complicating effects of earth's gravity and the interference from clinostat ambiguities. Using slow-speed centrifuges, coleoptiles received transversal accelerations in the hypogravity range between 0.1 and 1.0 g over periods that ranged from 2 to 130 min. All responses that occurred in weightlessness were compared to clinostal experiments on earth using the same apparatus.
Characteristic gravitropistic response patterns of Avena were not substantially different from those observed in ground-based experiments. Gravitropic presentation times were extrapolated. The threshold at 1.0 g was less than 1 min (shortest stimulation time 2 min), in agreement with values obtained on the ground. The least stimulus tested, 0.1 g for 130 min, produced a significant response. Therefore the absolute threshold for a gravitropic response is less than 0.1 g.     

Effect of Peeling on IAA-induced Growth in Avena Coleoptiles

The act of peeling removes the epidermis exclusively from Avenacoleoptiles. Peeling inhibits IAA-induced growth, by inhibitingthe growth of segments incubated in the presence of IAA, andpromoting that of those incubated in water. The magnitude ofthe inhibition of IAA-induced growth is proportional to theamount of epidermis removed. It is shown that neither lateralswelling, wounding, anaerobiosis, nor exposure to supraoptimalconcentrations of IAA cause the inhibition. It is concludedthat in Avena coleoptiles the epidermis regulates the rate ofexpansion of the underlying parenchyma cells and is the principaltarget of IAA-action. Avena sativa L., oat, coleoptile, indol-3-ylacetic acid, auxin, extension growth     

Photogravitropic equilibrium in Avena coleoptiles: fluence rate-response relationships and dependence on dark adaptation and clinostating

Phototropism of Avena coleoptiles was measured in response to blue-light irradiation lasting between 2 and 24 h. During this time the coleoptiles established a bending angle of photogravitropic equilibrium that was dependent on the time of irradiation and also on the pretreatment in light or darkness prior to stimulation. The absolute threshold for the photogravitropic equilibrium in response to blue light was 10(-8) micromol m(-2) s(-1). Photon fluence rate-response curves, which were generated after several hours of dark adaptation, had a characteristic shape with a prominent optimum in the middle of the dynamic range. Curves which were generated without prior dark adaptation displayed no such optimum. Clinostating dark-adapted coleoptiles caused an increase of sensitivity and responsiveness during a 2-h period of unilateral irradiation. The advantages and the drawbacks of long-term irradiation experiments for the investigation of phototropism and the generation of action spectra are discussed.     

Coleoptiles are gravi-guiding systems vital for gravi-insensitive shoots of germinating grass seedlings

Surgical removal of the coleoptiles from germinating seedlings of Secale cereale L., Avena sativa L., and Zea mays L. leads to a total loss of gravitropic response of the shoot without affecting growth. The principal function of coleoptiles is therefore to guide the gravi-insensitive shoot to the soil surface.Dedicated to Prof. Andreas Sievers on the occasion of his retirement     

Diferulic and ferulic acid in the cell wall of Avena coleoptiles—Their relationships to mechanical properties of the cell wall

Alkaline hydrolysis liberated ferulic and diferulic acid from polysaccharides of the Avena coleoptile ( Avena sativa L. cv. Victory I) cell walls. The amount of the two phenolic acids bound to cell walls increased substantially at day 4–5 after sowing, when the growth rate of the coleoptile started to decrease. The level of these acids was almost constant from the tip to base in 3-day-old coleoptiles, but increased toward the basal zone in 4- and 5-day-old ones. The ratio of diferulic acid to ferulic acid was almost constant irrespective of coleoptile age and zone. An increase in the amount of ferulic and diferulic acids bound to cell wall polysaccharides correlated with a decrease in extensibility and with an increase in minimum stress-relaxation time and relaxation rate of the cell wall. The level of lignin in the cellulose fraction increased as coleoptiles aged, but this increase did not correlate with changes in mechanical properties of the cell walls. These results suggest that ferulic acid, ester-linked to cell wall polysaccharides, is oxidized to give diferulic acid, which makes the cell wall mechanically rigid by cross-linking matrix polysaccharides and results in limited cell extension growth. In addition, it is probable that the step of feruloylation of cell wall polysaccharides is rate-limiting in the formation of in-termolecular bridges by diferulic acid in Avena coleoptile cell walls.     

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.     

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.     

A gas chromatographic analysis of the release of arabinose from coleoptile cell walls under the influence of auxin and dextranase preparations

An analysis by gas chromatography of the products of digestion of cell wall pellets of Avena sativa L., coleoptiles shows that after short term autolysis of pellets from auxin-rich plants, arabinose is released. Addition of arabinan as a substrate increases the yield of arabinose considerably, suggesting that a similar substance is broken down in the walls.In pellets from auxin-poor coleoptiles much less arabinose is released and addition of arabinan has no effect. Dextranase preparations stimulate this release and can also break down arabinan. This suggests that auxin and a dextranase-like enzyme are involved in the process.     

Effects of Exogenously Applied Jasmonates on Growth and Intracellular pH in Maize Coleoptile Segments

The effects of jasmonic acid (JA) on elongation growth of coleoptile segments from etiolated maize (Zea mays L.) were investigated in the presence and absence of auxin. When supplied alone, at physiological concentrations (10⁻⁹, 10⁻⁸, and 10⁻⁵ m), JA (or methyl-JA) inhibited growth. JA at a similar range of concentrations also inhibited auxin-induced elongation growth. To determine whether this effect on growth depended on endogenous abscisic acid (ABA), we grew maize coleoptiles in the presence of norflurazon (an inhibitor of carotenoid biosynthesis) that results in reduced endogenous ABA levels. Growth of etiolated coleoptile segments from these plants was inhibited by JA (or methyl-JA) in both the absence and presence of auxin. Previously, we have observed a correlation between elongation growth and cytosolic pH (pH_i), in which auxin lowers pH_i, and growth inhibitors such as ABA raise pH_i. We examined the effect of low concentrations of methyl-JA on pH_i with dual emission dye, carboxy seminaphthorhodafluor-1, and confocal microscopy. To confirm these studies, we also used in vivo ³¹P NMR spectrometry to ascertain the changes in pH_i after addition of jasmonate to maize coleoptiles. Coleoptiles grown in either the absence or presence of norflurazon responded to methyl-JA or JA by increases in pH_i of approximately 0.2 pH unit. This response occurs over a period of 15–20 min and appears to be independent of endogenous ABA. This alkalization induced by JA is likely to form a permissive environment for JA signal transduction pathway(s). Received February 5, 1999; accepted August 25, 1999     

Inhibition of selected plant systems by stereoisomers of chloramphenicol

The stereospecificity of chloramphenicol isomers on the inhibition of several plant systems was investigated. l-Threo, d-erythro, l-erythro and the antibiotic d-threo-chloramphenicol were effective inhibitors of auxin-induced elongation, ¹⁴C-leucine uptake and ¹⁴C-leucine incorporation into the protein fraction of coleoptiles from Avena sativa and Triticum vulgare. The isomers also inhibited Avena coleoptile uptake of ¹⁴C-α-aminoisobutyric acid and the de novo synthesis of α-amylase by aleurone layers from Hordeum vulgare seeds. All four compounds inhibited these processes to about the same extent and over a similar high concentration range (5 × 10⁻⁴ to 5 × 10⁻³ M). Bioassay of extracts from Avena coleoptiles treated with the non-antibiotic isomers revealed no tissue conversion into d-threo-chloramphenicol.     

The effect of the seed coat, embryonic axis and aeration conditions on starch degradation hi cotyledons of Lens culinaris

The effect of exogenously applied galactose on the cell wall polysaccharide synthesis and UDP-sugar levels in oat ( Avena sativa L. cv. Victory I) coleoptile segments was studied to clarify the mechanism of inhibition of IAA-induced cell elongation by galactose, and the following results were obtained: (1) The inhibition of IAA-induced cell elongation by galactose became apparent after a 2 h-lag, while the lag was shortened to 1 h when galactose was added to the segments after more than 1 h of IAA application. (2) Galactose inhibited the [¹⁴C]-glucose incorporation into cellulosic and non-cellulosic fractions of the cell wall and the increase in net polysaccharide content in the fractions during long-term incubation. (3) The dominant sugar nucleotide in oat coleoptiles was UDP-glucose (2.1 nmol segment⁻¹). Galactose application caused a remarkable decrease in the UDP-glucose level, accompanying a strong accumulation of galactose-1-phosphate and UDP-galactose. (4) Galactose-1-phosphate competitively inhibited the UTP: a- d -glucose-1-phosphate uridylyltransferase (EC 2.7.7.9) activity of the crude enzyme preparation from oat coleoptiles. From these results we conclude that galactose inhibits the IAA-induced cell elongation by inhibiting the formation of UDP-glucose, which is a key intermediate of cell wall polysaccharide synthesis.