Identification of tryptophan in leachate of oat hulls (Avena sativa) as a mediator of root growth regulation

Leachate of oat hulls ( Avena sativa L. cv. Sang) inhibits root elongation and causes swollen roots and abundant root hair formation. The active substance was isolated by column chromatography and thin layer chromatography (TLC) systems. High performance liquid chromatography (HPLC) revealed 3 peaks, one of which corresponded to the substance responsible. The latter was identified as tryptophan by means of its UV spectrum, amino acid analysis, nuclear magnetic resonance spectrometry (NMR) and mass spectrometry (MS).     

Auxin activity of 3-hydroxymethyl oxindole and 3-methylene oxindole in oat

Two photooxidation products of indol-3-ylacetic acid (IAA), 3-hydroxymethyl oxindole (HMO) and 3-methylene oxindole (Meox) were almost as effective as IAA in stimulation of growth of oat (Avena saliva cv. Kent) coleoptile sections. The IAA failed to give stimulation of growth after pretreatment of the coleoptiles with chemicals inhibiting oxidation of IAA, but the growth promoting effects of HMO or Meox remained unaffected by such pretreatment. After pretreatment with the chemicals which can form an adduct with Meox, the growth promoting activities of IAA, HMO or Meox were lost showing the involvement of the oxindole pathway of IAA metabolism in IAA action. Time-course experiments also showed that the stimulatory effects of HMO and Meox were the same as IAA.     

The antagonism of IAA-induced hydrogen ion extrusion and coleoptile growth by diclofop-methyl

Diclofop-methyl (DM) (ester) was readily absorbed by peeled and unpeeled coleoptiles of wheat, Triticum aestivum L. cv. Waldron, and oat, Avena sativa L. cv. Garry. Substantial absorption of diclofop (acid) occurred only in peeled coleoptiles of the two species. IAA-induced acidification in peeled coleoptiles of both species was inhibited by 100 μ M DM or diclofop (acid) during a 3 to 4 h period. There was no recovery of acidification after DM or diclofop inhibition in oat coleoptiles; however, acidification in wheat coleoptiles recovered from inhibition by DM but not from diclofop. The recovery from DM inhibition may be due to a reduction in the diclofop pool derived from DM by efflux and metabolism (detoxification) in peeled wheat coleoptiles. Diclofop was not detoxified in oat coleoptiles. IAA-induced elongation of unpeeled oat coleoptiles was inhibited totally by 100 μ M DM but not by 100 μ M diclofop after 3.3 h of treatment. Wheat coleoptile elongation was relatively unaffected by either DM or diclofop. Basal elongation (no IAA) of both wheat and oat coleoptiles was inhibited by DM and diclofop. The inhibition by DM appeared to be irreversible, whereas the inhibition by diclofop was overcome by the addition of 10 μ M IAA.     

Comparison of K,MgATPases in purified plasmalemma from wheat and oat. – Substrate specificities and effects of pH, temperature and inhibitors

Plasmalemma from 8-day old oat ( Avena sativa L. cv. Brighton) and spring wheat ( Triticum aestivum L. cv. Drabant), grown in the dark at 18°C, was prepared from the 10000 g (10 min) – 30 000 g (60 min) root homogenate by two-phase separation in three steps with 6.5% (w/w) Dextran T 500 and 6.5% (w/w) polyethylene glycol 4 000. Biochemically and with respect to activation by Mg²⁺ as well as by (Mg²⁺+ K⁺), the oat preparations clearly appeared as ATPase(s) in the pH range 5–8. They showed high specificity for ATP, temperature optima between 38 and 40°C, and were inhibited by vanadate, DCCD (dicyclohexylcarbodiimide) and SH-reagents, but not by oligomycin, ammonium molybdate or ouabain. In contrast, the preparations from wheat contained more than one type of MgATPase/ nucleotidase, as revealed by complex dependence on both pH and temperature as well as by comparatively low specificity towards nucleotides. However, no unspecific phosphatase was present, and the effect of K⁺ over and above that of Mg²⁺ was almost as specific as in oat by all criteria used. The data available from this and earlier investigations from our group would indicate that the complex reactions of preparations of wheat plasmalemma may not be due to contamination but, rather, expressions of the many biological functions that must be associated with the plasmalemma in vivo and which may be located in sub-units that are more firmly attached to wheat than to oat plasmalemma.     

Inhibition of the synthesis of cell wall polysaccharides in oat coleoptile segments by jasmonic acid: Relevance to its growth inhibition

The inhibitory mode of action of jasmonic acid (JA) on the growth of etiolated oat (Avena sativa L. cv. Victory) coleoptile segments was studied in relation to the synthesis of cell wall polysaccharides using [¹⁴C]glucose. Exogenously applied JA significantly inhibited indoleacetic acid (IAA)-induced elongation of oat coleoptile segments and prevented the increase of the total amounts of cell wall polysaccharides in both the noncellulosic and cellulosic fractions during coleoptile growth. JA had no effect on neutral sugar compositions of hemicellulosic polysaccharides but substantially inhibited the IAA-stimulated incorporation of [¹⁴C]glucose into noncellulosic and cellulosic polysaccharides. JA-induced inhibition of growth was completely prevented by pretreating segments with 30 mm sucrose for 4 h before the addition of IAA. The endogenous levels of UDP-sugars, which are key intermediates for the synthesis of cell wall polysaccharides, were not reduced significantly by JA. Although these observations suggest that the inhibitory mode of action of JA associated with the growth of oat coleoptile segments is relevant to sugar metabolism during cell wall polysaccharide synthesis, the precise site of inhibition remains to be investigated.Abbreviations JA jasmonic acid - ABA abscisic acid - IAA indoleacetic acid - T ₀ minimum stress relaxation time - TFA trifluoroacetic acid - TCA trichloroacetic acid - HPLC high-performance liquid chromatography - EtOAc ethyl acetate - TLC thin-layer chromatography - JA-Me methyl jasmonate - GLC-SIM gas-liquid chromatography-selected ion monitoring     

Isoelectric focusing of oat root membranes

The feasibility of purifying subcellular membranes, especially plasma membranes, from oat roots using isoelectric focusing has been examined. Membranes from oat (Avena sativa L. cv Garry) root homogenates were fractionated using discontinuous sucrose density gradient centrifugation and then electrofocused using a microanalytical isoelectric focusing column. The column contained either a broad-range (pH 3-10) or narrow-range (pH 3-6) pH gradient stabilized by a 5 to 15% Ficoll gradient. Results from the broad-range columns confirmed that the isoelectric pH (pI) values of the membranes were in the acidic range, with pI values ranging from 3.9 to 5.2. Using narrow-range pH gradients, it was possible to fractionate further plasma membrane-enriched material obtained from a sucrose density gradient. We had no success at fractionating crude membrane preparations from oat roots. Narrow-range pH gradients generated by commercial ampholytes were more successful than those generated by acetate/acetic acid mixtures.     

A molecular linkage map of cultivated oat.

A molecular linkage map of cultivated oat composed of 561 loci has been developed using 71 recombinant inbred lines from a cross between Avena byzantina cv. Kanota and A. sativa cv. Ogle. The loci are mainly restriction fragment length polymorphisms detected by oat cDNA clones from leaf, endosperm, and root tissue, as well as by barley leaf cDNA clones. The loci form 38 linkage groups ranging in size from 0.0 to 122.1 cM (mean, 39 cM) and consist of 2-51 loci each (mean, 14). Twenty-nine loci remain unlinked. The current map size is 1482 cM and the total size, on the basis of the number of unlinked loci, is estimated to be 2932.0 cM. This indicates that this map covers at least 50% of the cultivated oat genome. Comparisons with an A-genome diploid oat map and between linkage groups exhibiting homoeology to each other indicate that several major chromosomal rearrangements exist in cultivated oat. This map provides a tool for marker-assisted selection, quantitative trait loci analyses, and studies of genome organization in oat.     

Differential responses of oat cultivars to phosphate deprivation: plant growth and acid phosphatase activities

The effect of phosphate starvation on growth and acid phosphatases (APases) localization and activity in oat tissues was investigated. Oat cultivars (Avena sativa L.??Arab, Polar, Szakal) were grown for 1?C3?weeks in complete nutrient medium (+P) and without phosphate (?P). Pi concentration in plant tissues decreased strongly after culturing on ?P medium. Pi deficit reduced shoot growth, stimulated root elongation and increased ratio of root/shoot in all oat cultivars. Pi deficit had a greater impact on growth of oat cv. Polar than other varieties. A decrease in the internal Pi status led to an increase of acid phosphatase activities in extracts from shoots and roots, and in root exudates. The highest activity of secreted APases was observed for oat cv. Arab, during the third week of growth under Pi-deficient conditions. The activity of extracellular APase was high in young, growing zones of roots of ?P plants. Histochemical visualization indicated high activity of APases in the epidermis and vascular tissues of ?P plants. Pi deficiency increased intracellular APase activity in shoot mainly in oat cv. Polar, whereas APase activity in roots was the highest in oat cv. Szakal. Protein extracts from roots and shoots were run on native discontinuous PAGE to determine which isoform(s) may be affected by Pi deficiency. Three major APase isoforms were detected in all oat plants; one was strongly induced by Pi deficit. The studied oat cultivars differed in terms of acclimation to deficiency of phosphate??used various pools of APases to acquire Pi from external or internal sources.     

Physiological responses of resistant and susceptible plants to diclofop-methyl and its antagonism by 2,4-D and antioxidants

Diclofop-methyl (DM) sprayed onto 6–8-week-old plants of leafy spurge ( Euphorbia esula L.) caused senescence and abscission of older leaves, while the young leaves and apex remained attached. The phytotoxicity of DM was reversed by the antioxidant, α -tocopherol (vitamin E), in leafy spurge and DM-susceptible oat ( Avena sativa L. cv. Gary). DM and 2,4-dichlorophenoxyacetic acid (2,4-D) increased ethylene evolution in mature leaves of leafy spurge. Vitamin E reduced the DM-induced ethylene by ampproximately 50%, but had no effect on the 2,4-D-induced ethylene. DM did not increase ethylene in DM-resistant pea or tobacco, but 2,4-D induced a 3-fold increase in ethylene evolution over controls in DM-resistant tobacco. 2,4-D amppears to act at a site different from that of DM in the pathway of ethylene formation. Ethylene evolution increased in DM-treated susceptible biotypes of annual ryegrass ( Lolium rigidum L.) and wild oat ( Avena fatua L.), but not in resistant biotypes of these species. DM reduced root and shoot formation and dry weight in hypocotyl segments of etiolated leafy spurge seedlings grown in vitro. Organogenesis and dry weights were increased by the combination of DM+antioxidants. Vitamin E was a more effective antioxidant than ascorbic acid. These results sumpport the hypothesis that DM induces oxidative stress in susceptible plant tissues and that antioxidants reduce the damaging action of the phytotoxic free radicals.     

Regulation of ethylene biosynthesis during senescence of oat leaf segments

The evolution of endogenous ethylene, the conversion of 1-aminocylopropane-1-car-boxylic acid (ACC) to ethylene and the amounts of ACC (free and conjugated) have been followed during the senescence of oat ( Avena sativa L. cv. Victory) leaf segments. During the first three days of incubation of leaf segments in darkness, endogenous ethylene evolution and ACC-dependent ethylene production displayed a close relationship, both showing an increase followed by a decrease to the basal rate. However, unlike ethylene production, the level of ACC increased during the five days of incubation in the dark without any decline. It is concluded that ACC synthesis does not limit ethylene production, at least in the last stages of leaf senescence when ethylene production markedly decreased. The level of conjugated ACC increased and reached a plateau already at the first day of incubation. Yet, at the progressive stages of senescence, when the level af ACC gradually increased, no further conjugation of ACC could be detected. Thus, conjugation of ACC cannot account for ethylene drop at the last stages of oat leaf senescence.     

Short term phytochrome control of oat coleoptile and pea epicotyl growth

Continuous recordings of the effect of light on oat (Avena sativa L. cv. Victory) coleoptile and pea (Pisum sativum L. cv. Alaska) epicotyl growth were made. Using a single excised coleoptile 10 minutes of red light was found to promote growth after a latent period of 46 minutes. The stimulation was transient and was not far red-reversible. Blue and far red light also promoted growth with similar kinetics. The action of continuous red or far red light was similar to that of 10-minute light. The growth of the intact pea third internode (as well as excised segments) was strongly inhibited by red light, with a latent period of 80 minutes. This effect was far red-reversible, and far red and blue light caused only a slight inhibition of growth.     

Purification of oat and rye phytochrome

A purification procedure employing normal chromatographic techniques is outlined for isolating phytochrome from etiolated oat (Avena sativa L.) seedlings. Yields in excess of 20% (25 milligrams or more) of phytochrome in crude extract were obtained from 10- to 15-kilograms lots. The purified oat phytochrome had an absorbance ratio (A₂₈₀ nm/A₆₆₅ nm) of 0.78 to 0.85, comparable to reported values, and gave a single major band with an estimated molecular weight of 62,000 on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. A modification of the oat isolation procedure was used to isolate phytochrome from etiolated rye Secale cereale cv. Balbo) seedlings. During isolation rye phytochrome exhibited chromatographic profiles differing from oat phytochrome on diethylaminoethyl cellulose and on molecular sieve gels. It eluted at a higher salt concentration on diethylaminoethyl cellulose and nearer the void volume on molecular sieve gels. Yields of 5 to 10% (7.5-10 milligrams) of phytochrome in crude extract were obtained from 10- to 12-kilogram seedling lots. The purified rye phytochrome had an absorbance ratio of 1.25 to 1.37, significantly lower than values in the literature and gave a single major band with an estimated molecular weight of 120,000 on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. It is suggested that the absorbance ratio and electrophoretic behavior of rye phytochrome are indices of purified native phytochrome, and that oat phytochrome as it has been described is an artifact which arises as a result of endogenous proteolysis during isolation. A rationale is provided for further modifications of the purification procedure to alleviate presumed protease contaminants.     

The role of calcium in growth induced by indole-3-acetic acid and gravity in the leaf-sheath pulvinus of oat (Avena sativa)

Leaf-sheath pulvini of excised segments from oat (Avena sativa L.) were induced to grow by treatment with 10 M indole-3-acetic acid (IAA), gravistimulation, or both, and the effects of calcium, EGTA, and calcium channel blockers on growth were evaluated. Unilaterally applied calcium (10 mM CaCl₂) significantly inhibited IAA-induced growth in upright pulvini but had no effect on growth induced by either gravity or gravity plus IAA. Calcium alone had no effect on upright pulvini. The calcium chelator EGTA alone (10 mM) stimulated growth in upright pulvini. However, EGTA had no effect on either IAA-or gravity-induced growth but slightly diminished growth in IAA-treated gravistimulated pulvini. The calcium channel blockers lanthanum chloride (25 mM), verapamil (2.5 mM), and nifedipine (2.5 mM) greatly inhibited growth as induced by IAA (50% inhibition) or IAA plus gravity (20% inhibition) but had no effect on gravistimulated pulvini. Combinations of channel blockers were similar in effect on IAA action as individual blockers. Since neither calcium ions nor EGTA significantly affected the graviresponse of pulvini, we conclude that apoplastic calcium is unimportant in leaf-sheath pulvinus gravitropism. The observation that calcium ions and calcium channel blockers inhibit IAA-induced growth, but have no effect on gravistimulated pulvini, further supports previous observations that gravistimulation alters the responsiveness of pulvini to IAA.     

A fluorescent compound in oat root plasma membrane

Homogenates of 7-day-old oat (Avena sativa L. cv. Brighton) roots were highly fluorescent (excitation and emission maxima around 360 and 440 nm, respectively). Less than ¹/₁₀ as much fluorescence per g fresh weight was found in oat shoots or in wheat (Triticum aestivum L. cv. Drabant) roots or shoots. Most of the fluorescence of oat roots was found in the soluble fraction (150 000g supernatant). However, some could be detected in the plasma membrane fraction (excitation and emission maxima 365 and 417 nm, respectively), which contained a 3-fold higher fluorescence per mg protein than the homogenate. Growth of oat or wheat in a medium containing, 10-^?5M scopoletin (6-methoxy-7-hy-droxy coumarin), a fluorescent compound previously reported to be present in both wheat and oat roots, caused the disappearance of scopoletin from the medium (proportional to the amount of roots) and the appearance of increased fluorescence in the root homogenates but not in the shoot homogenates. In both oat and wheat roots ail of the extra fluorescence was recovered in the soluble fraction and at least in wheat it consisted of unconverted scopoletin. The concentration of scopoletin in wheat roots grown in 10-^?5M scopoletin was around 50 nmol (g fresh weight)^?1, or about five times the concentration in the growth medium. Scopoletin in the growth medium (10-^?5M) or in the assays (up to 10-^?4M) did not affect Mg²⁺-, Mg²⁺+K⁺- or Ca²⁺-ATPase activities in wheat or oat roots. The fluorescence properties of the oat plasma membrane were different from those of authentic scopoletin. Either the surroundings modify the fluorescence of membrane-associated scopoletin or the endogenous fluorescent compound is not scopoletin but a glycoside-derivative of scopoletin or some completely unrelated compound.     

Regulation of root formation by auxin-ethylene interaction in pea stem cuttings

The possibility was investigated that the inhibition of rooting in pea ( Pisum sativum L. cv. Weibull's Marma) cuttings caused by low indol-3yl-acetic acid (IAA) concentrations is due to ethylene produced as a result of IAA treatment. Treatment with 10 uμ IAA reduced the number of roots to about 50% of the control and increased ethylene production in the stem bases by about 20 times the control value during the two first days of treatment. Ethylene-releasing compounds (ethephon and 1-amino-cyclopropane-1-carboxylic acid, ACC), in concentrations giving a similar ethylene release, inhibited rooting to the same extent or more strongly than IAA. These results indicate that IAA-induced ethylene is at least responsible for the negative component in IAA action on root formation in pea cuttings. A higher IAA concentration (100 μ) and indol-3yl-butyric acid efficiently counteracted the negative effect of ethylene on root formation.     

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.     

Wide hybridization between oat and pearl millet belonging to different subfamilies of Poaceae

Oat (Avena sativa L.) and pearl millet (Pennisetum glaucum L.) belong to different subfamilies of Poaceae. When emasculated oat was pollinated by millet, fertilization took place and all seven millet chromosomes were retained along the complete haploid oat complement during early stages of embryogenesis. Fourteen days after pollination, we cultured 170 embryos onto rescue medium, of which 99 were attached with endosperm tissue. Twenty-one embryos germinated and showed shoot growth. One of them also developed roots. The shoots of the rootless embryos elongated, but rolled to the scutellum side and eventually died in light conditions. Chromosome observations and marker analyses indicated that the seedling plants were true hybrids that retained all of the oat and millet chromosomes. One exceptional embryo with shoot and root grew under light conditions. This was a haploid of oat and developed to a fertile adult plant. One embryo generated a callus after 6 months cultivation, and it was found to harbor four out of the seven millet chromosomes corresponding to linkage groups 2, 4, 6, and 7. The callus grew vigorously but did not develop shoots or roots.     

Effects of salicylhydroxamate on respiration, seed germination and seedling growth in Avena fatua

The effects of inhibitors of alternative respiration [salicylhydroxamate (SHAM) and propyl gallate (PG)] on germination, seedling growth and O₂ uptake in Avena fatua L. (wild oats) were studied. SHAM did not inhibit germination or O₂ uptake prior to germination. SHAM-sensitive (alternative) respiration, therefore, cannot be a pre-requisite for germination. Following germination, both chemicals inhibited seedling growth with the root being more susceptible than the shoot. SHAM concentrations that inhibited root growth by 90 to 95%, inhibited O₂ uptake of 1 cm root apices by less than 15%. While sodium azide (a cytochrome-oxidase inhibitor; 1 m M ) alone inhibited O₂ uptake by only 40 to 50%, in the simultaneous presence of SHAM (or PG), O₂ uptake was inhibited by 90 to 99%. Thus: 1) respiration of wild oat seedling root apices is predominantly cytochrome-mediated and incomplete inhibition of O₂ uptake in the presence of azide alone is due to diversion of electrons to the alternative pathway and 2) even though these roots have little alternative respiration, they maintain the capacity to support a much greater flux of electrons via this path way. SHAM and PG at concentrations (0.05 to 0.4 m M ) which inhibited O₂ uptake significantly in the presence (but not in the absence) of azide had little effect on root growth suggesting that an effect(s) other than that on respiration is involved in the inhibition of root growth at higher concentrations. The effect of SHAM on wild oat root growth is not selective as it also inhibits growth of a number of crop species.     

Molecular forms of arginine decarboxylase in oat leaves

Arginine decarboxylase (ADC, EC 4.1.1.19) is the first enzyme in one of the two biosynthetic pathways of putrescine in plants and has been characterized from a number of species, beginning with the work of Smith (1979; Phytochemistry 18: 1447–1452) who suggested that oat ADC had native sizes of 118 and 195 kDa. There are several studies showing a lack of correlation between changes in enzyme activity and mRNA or protein levels. In oats ( Avena sativa L.) a posttranslational modification of ADC has been described. The protein is synthesized as a 66-kDa precursor that is proteolytically processed into two polypeptides of 42 and 24 kDa with an associated gain of enzyme activity. In the present work, we have studied the existence of different ADC molecular forms in oat leaves by determination of enzymatic activity and using polyclonal antibodies obtained against an amino acid sequence near the C terminus deduced from the nucleotide sequence. In Avena sativa L. cv. Victory, we demonstrate the existence of 5 different molecular forms of ADC, with approximate molecular mass of 195, 115, 66, 38 and 23 kDa, that react with our antibodies and have enzymatic activity. Our results agree with previous work, but this is the first report showing all molecular forms simultaneously and might be a preliminary contribution to solve the question about the distribution of multiple forms of ADC and their importance in the correlation between the enzymatic activity and mRNA levels.     

Physiological mechanism of the auxin-induced increase in light sensitivity of phytochrome-mediated growth responses in Avena coleoptile sections

The physiology of the auxin-induced 10,000-fold increase in light sensitivity of a phytochrome-mediated growth response (Shinkle and Briggs, 1984 Proc Natl Acad Sci USA 81: 3742-3746) has been characterized in subapical coleoptile sections from dark-grown oat (Avena sativa L. cv Lodi) seedlings. Six micromolar indole-3-acetic acid (IAA) must be present for 1 hour before to 2 hour after irradiation in order to confer maximal sensitivity to light. The direct effect of IAA on growth can be separated from its effect on light sensitivity. Several classes of synthetic auxins will substitute for IAA in inducing an increase in sensitivity to light, as will both the phytotoxin fusicoccin and treatment of sections with pH 4.5 buffer. The increase in sensitivity to light induced by 6 micromolar IAA is completely inhibited by buffering the sections at pH 5.9 with 30 millimolar 2-(N-morpholino)ethanesulfonic acid. These findings suggest that the capacity to respond to very low fluences of light is regulated by extracellular pH.