Indoleacetic Acid biosynthesis in Avena coleoptile tips and excised bean shoots

Avena coleoptiles did not elongate when incubated with tryptophan under sterile conditions. Indole, anthranilic acid, and tryptamine promoted elongation. Under the same conditions, the tissue converted tryptophan-¹⁴C to IAA-¹⁴C. More IAA-¹⁴C was produced from indole-¹⁴C than from tryptophan-¹⁴C; however, the free tryptophan content of the tissue was also greatly increased by the indole treatment. Tryptophan-¹⁴C was readily taken up by the tissue but was mainly incorporated into protein and did not increase the free tryptophan level. When bean shoots were labeled with tryptophan-¹⁴C or indole-¹⁴C, the label incorporation into IAA-¹⁴C was very nearly the same. In this tissue the free tryptophan level in the tryptophan-¹⁴C and indole-¹⁴C treatments was also about equal. These results suggest that failure of exogenously supplied tryptophan to promote the elongation of Avena coleoptiles is a result of its predominant incorporation into protein and consequent unavailability for conversion to IAA.     

Chloride accumulation by mung bean root tips: a low affinity active transport system at the plasmalemma

Net uptake of Cl⁻ into root tips of mung bean (Phaseolus aureus) increases steadily with increasing external concentrations from 1 to 60 mm. Membrane potentials were measured to determine the equilibrium concentration of Cl⁻ in the tissue which could be due to diffusion. This concentration was readily exceeded in both the relatively nonvacuolate tips (0 to 1 mm) and the vacuolate, mature upper sectons (1 to 11 mm) of the roots. The activity coefficient of both cytoplasmic and vacuolar Cl⁻, measured with Cl⁻ sensitive microelectrodes, was approximately the same as that of a pure KCl solution of the same concentration. It is concluded that the “second mechanism” of ion uptake involves a large increase in the rate of active transport at the plasmalemma as the external concentration is increased above 1 mm.     

Effect of abscisic Acid on root hydraulic conductivity

Reports of the effects of abscisic acid (ABA) on ion and water fluxes have been contradictory. Some of the confusion seems due to the interaction of ion and water transport across membranes. In these experiments root systems were subjected to hydrostatic pressures up to 5.0 bars to enable measurement of root conductance that was independent of measurement of osmotic potentials or ion fluxes.     

Polyamines and root formation in mung bean hypocotyl cuttings : I. Effects of exogenous compounds and changes in endogenous polyamine content

The effect of several polyamines (putrescine, spermidine, and spermine), their precursors (l-arginine and l-ornithine), and some analogs and metabolic inhibitors (l-canavanine, l-canaline, and methylglyoxal-bis [guanylhydrazone]) on root formation have been studied in mung bean (Vigna radiata [L.] Wilczek) hypocotyl cuttings.     

Indoleacetic Acid and Abscisic Acid Antagonism: II. On the Phytochrome-Mediated Attachment of Barley Root Tips on Glass

The phytochrome-mediated attachment of mung bean (Phaseolus vulgaris L., var. Oklahoma 612) root tips on glass is quickly affected by indoleactic acid and abscisic acid at concentrations of 10 nm or less. Indoleacetic acid induces detachment, whereas abscisic acid induces attachment. Both plant regulators rapidly antagonize the action of the other. None of several cytokinins, gibberellins, and ethylene tested over a wide range in concentration had any effect on either attachment or detachment of root tips. It is postulated that phytochrome could control the endogenous levels of indoleacetic acid and abscisic acid and perhaps other hormones under certain circumstances, that this action is the first process initiated by phytochrome, and that indoleacetic acid and abscisic acid act on the plasmalemma to bring about opposing changes in the surface electric charges of plant cells.     

Influence of ethylene on adventitious root formation in mung bean hypocotyl cuttings

The relationship between ethylene and adventitious root formation in mung bean hypocotyl cuttings was studied.Ethephon, an ethylene-releasing compound, at 5 x 10 -5 M increased root number and root dry weight on hypo-cotyl cuttings. When ethephon was applied to hypocotyl at different times after excision, there were two effectivetimes for root production i.e. between 06 h and 18-24 h. These two time periods correspond to the induction phase and the late initiation phase of root development, respectively. After excision, three peaks of ethylene productionwere observed. The first peak commencing at 6 h started the sequence of reactions leading root formation, the second peak appearing at 12 h coincided with the beginning of the increase of the IAA level during primordia initiation, and the third peak showing at 48 h played a role in root differentiation and growth. Ethylene stimulated rooting by enhancing the increase in auxins. Thus it appears that the IAA-induced ethylene production may be a factor involved in the stimulation of adventitious root formation.     

Effect of indoleacetic acid,abscisic acid,root tips and coleoptile tips on growth and curvature of maize roots

The effect of externally applied indoleacetic acid (IAA) and abscisic acid (ABA) on the growth of roots of Zea mays L. was measured. Donor blocks of agar with IAA or ABA were placed laterally on the roots and root curvature was measured. When IAA was applied to vertical roots, a curvature directed toward the donor block was observed. This curvature corresponded to a growth inhibition at the side of the root where the donor was applied. When IAA was applied to horizontal roots from the upper side, normal geotropic downward bending was delayed or totally inhibited. The extent of retardation and the inhibition of curvature were found to depend on the concentration of IAA in the donor block. ABA neither induced curvature in vertical roots nor inhibited geotropic curvature in horizontal roots; thus the growth of roots was not inhibited by ABA. However, when, instead of donor blocks, root tips or coleoptile tips were placed onto vertical roots, a curvature of the roots was observed.Abbreviations ABA abscisic acid - IAA 3-indoleacetic acid     

Purification and properties of glucosidase I from mung bean seedlings

The microsomal enzyme fraction from mung bean seedlings was found to contain glucosidase activity capable of releasing [3H]glucose from the glucose-labeled Glc3Man9GlcNAc. The enzymatic activity could be released in a soluble form by treating the microsomal particles with 1.5% Triton X-100. When the solubilized enzyme fraction was chromatographed on DE-52, it was possible to resolve glucosidase I activity (measured by the release of [3H]glucose from Glc3Man9GlcNAc) from glucosidase II (measured by release of [3H]glucose from Glc2Man9GlcNAc). The glucosidase I was purified about 200-fold by chromatography on hydroxylapatite, Sephadex G-200, dextran-Sepharose, and concanavalin A-Sepharose. The purified enzyme was free of glucosidase II and aryl-glucosidase activities. Only a single glucose residue could be released from the Glc3Man9GlcNAc by this purified enzyme and the other product was the Glc2Man9GlcNAc. Furthermore, this enzyme was inhibited in a dose-dependent manner by kojibiose, an alpha-1,2-linked glucose disaccharide, but not by other alpha-linked glucose disaccharides. These data indicate that this glucosidase is a specific alpha-1,2-glucosidase. The pH optimum for the glucosidase I was about 6.3 to 6.5, and no requirements for divalent cations were observed. The enzyme was inhibited strongly by the glucosidase processing inhibitors, castanospermine and deoxynojirimycin, and less strongly by the plant pyrrolidine alkaloid, 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine. However, the enzyme was not inhibited by the mannosidase processing inhibitors, swainsonine, deoxymannojirimycin or 1,4-dideoxy-1,4-imino-D-mannitol. The stability of the enzyme under various conditions and other properties of the enzyme were determined.     

Ion fluxes and phytochrome protons in mung bean hypocotyl segments: I. Fluxes of potassium

K⁺ [⁸⁶Rb⁺] uptake by Phaseolus aureus Roxb. hypocotyl segments cut immediately below the hook is inhibited by the active form of phytochrome (Pfr). Short load-short wash experiments indicate that the inhibition of uptake occurs across the plasmalemma. A maximal inhibition of short term uptake occurs in 10 to 50 millimolar KCI. Low temperature had only a small effect on influx and the inhibition of influx from 50 millimolar KCI. A consideration of the electrochemical gradient for K⁺ suggests that passive K⁺ fluxes may predominate under these conditions. Red light induces small depolarizations of membrane potential in subhook cells. Far red light antagonizes this effect. Pfr inhibits efflux of K⁺[⁸⁶Rb⁺] from subhook segments. This effect is also relatively insensitive to low temperature. This inhibition of efflux may reflect inhibition of a K⁺ -K⁺ exchange process, or reduced passive permeability of the plasmalemma to K⁺. In contrast, Pfr enhances short term uptake of K⁺[⁸⁶Rb⁺] in apical hypocotyl hook segments of Phaseolus aureus Roxb. Short load-short wash experiments indicate that fluxes across the plasmalemma are modified by Pfr. A maximal enhancement of short term influx occurs in 50 millimolar KCI. Influx and the red light enhancement of influx from 50 millimolar KCI are relatively insensitive to low temperature. Pfr also enhances efflux of K⁺[⁸⁶Rb⁺] from preloaded apical hook segments. This increased influx may reflect enhancement of a K⁺ -K⁺ exchange process or increased passive permeability of the plasmalemma to K⁺.     

Effects of cycloheximide on abscisic Acid biosynthesis and stomatal aperture in bean leaves

Cycloheximide was shown to block abscisic acid (ABA) biosynthesis in nonstressed as well as in stressed Phaseolus vulgaris leaves. Leaf wilting caused by cycloheximide resulted from increased stomatal opening as judged by a decreased stomatal diffusion resistance. The inhibition of ABA biosynthesis by cycloheximide was at least partially responsible for the increase in stomatal opening as suggested by the cooccurrence of inhibition of ABA biosynthesis and increased stomatal opening, and the partial reversal of stomatal opening in cycloheximide-treated leaves by exogenous ABA. Dark treatment failed to close stomatal in cycloheximide-treated leaves, suggesting that stomatal closure in response to darkness may normally be mediated by ABA.     

The effects of abscisic Acid on growth and nucleic Acid synthesis in excised embryonic bean axes

Abscisic acid (ABA) is an effective inhibitor of cell elongation in excised embryonic bean axes whether added prior to or after the initiation of cell elongation. Zeatin partially reverses this growth inhibition. ABA inhibits ³²P incorporation into ribosomal RNA, transfer RNA, and DNA but not into the tenaciously bound fraction of elongating axes in a manner resembling 5-fluorouracil, a compound which does not inhibit axis growth. The methylated albumin on kie-selguhr elution profiles of nucleic acids obtained from axes treated with either ABA, 5-fluorouracil, or a combination of the two are similar, and zeatin treatment has little apparent effect on these results. Our results suggest that the inhibition of growth in the axes by ABA is not due to its inhibition of DNA synthesis.     

Correlation between extracellular fibrils and attachment of Rhizobium leguminosarum to pea root hair tips.

As part of a project meant to characterize molecules involved in nodulation, a semiquantitative microscopic assay was developed for measuring attachment of Rhizobium leguminosarum cells to pea root hair tips, i.e., the site at which R. leguminosarum initiates nodulation. This form of attachment, designated as cap formation, was dependent on the incubation pH and growth phase, with optimal attachment at pH 7.5 and with bacteria in the early stationary phase of growth. Addition of glucose to the growth medium delayed the initiation of the stationary phase and cap formation, suggesting a correlation between cap formation and carbon limitation. Attachment of R. leguminosarum was not inhibited by pea lectin haptens which makes it unlikely that lectins are involved under the tested conditions. Moreover, heterologous fast-growing rhizobia adhered equally well to pea root hair tips. Since the attachment characteristics of a Sym plasmid-cured derivative were indistinguishable from those of the wild-type strain, the Sym plasmidborne nodulation genes are not necessary for attachment. Sodium chloride and various other salts abolished attachment when present during the attachment assay in final concentrations of 100 mM. R. leguminosarum produced extracellular fibrils. A positive correlation between the percentage of fibrillated cells and the ability of the bacteria to form caps and to adhere to glass and erythrocytes was observed under various conditions, suggesting that these fibrils play a role in attachment of the bacteria to pea root hair tips, to glass, and to erythrocytes.     

Effect of membrane surface charge on nickel uptake by purified mung bean root protoplasts

The influence of membrane surface charge on cation uptake was investigated in protoplasts prepared from roots of mung bean (Vigna radiata L.). Confocal laser scanning microscopy showed that a fluorescent trivalent cation accumulated to very high concentrations at the surface of the protoplasts when they were incubated in medium containing low concentrations of Ca or other cations, but that this accumulation could be completely reversed by suppression of membrane surface negativity by high cation concentrations. Influx of 63Ni was strongly reduced by a range of divalent cations. Increasing the Ca concentration in the medium from 25 microM to 10 mM inhibited 63Ni influx by more than 85%. 63Ni influx was also inhibited by 85% by reducing the pH from 7 to 4. Computation of the activity of Ni at the membrane surface under the various treatment conditions showed that Ni uptake was closely correlated with its activity at the membrane surface but not with its concentration in the bulk medium. It was concluded that the effects on Ni uptake of addition of monovalent, divalent and trivalent cations, and of variations in pH are all consistent with the proposition that the activity of Ni at the membrane surface is the major determinant of the rate of Ni influx into mung bean protoplasts. It is proposed that the surface charge on the plasma membrane will influence the membrane transport of most charged molecules into cells.     

Effect of auxins and plant oligosaccharides on root formation and elongation growth of mung bean hypocotyls

The interaction of auxins – IAA, IBA or NAA – with galactoglucomannan oligosaccharides (GGMOs) on adventitious root formation and elongation growth of mung bean hypocotyl cuttings was studied. GGMOs induced adventitious roots in the absence of auxins; however, their effect was lower compared with IBA or NAA. On the other hand, in the presence of auxins, GGMOs inhibited adventitious root induction. Their effect depended on the concentration of oligosaccharides and the type of auxin used. The highest inhibition effect of GGMOs at a concentration of 10⁻⁸ M in the presence of IBA and NAA was observed. In the presence of IAA their inhibition was non-significant in regard to the concentration. The interaction of auxins with GGMOs resulted in the formation of adventitious roots on a shorter part of hypocotyls compared with the effect of auxins alone. However, roots were induced more extensively along the hypocotyls treated with GGMOs compared with the control. GGMOs inhibited the length of induced adventitious roots in the presence of IAA, while in combination with IBA or NAA they were ineffective. The elongation of hypocotyls induced by IAA or IBA was inhibited by GGMOs, too. However, in the presence of NAA or by endogenous growth they were without any significant effect on elongation growth. These findings suggest that GGMOs in certain concentrations might inhibit rooting and the elongation process dependant on auxin used.     

Cholinesterases from plant tissues: I. Purification and characterization of a cholinesterase from mung bean roots

A cholinesterase was purified 36-fold from mung bean (Phaseolus aureus) roots by a combination of differential extraction media and gel filtration. The enzyme could be effectively extracted only by high salt concentration, indicating that it is probably membrane-bound. Methods used for assaying animal cholinesterases were tested, two of which were adapted for use with the bean cholinesterase. The bean enzyme hydrolyzed choline and noncholine esters but showed its highest affinity for acetylcholine and acetylthiocholine. The pH optimum was 8.5 for acetylthiocholine and 8.7 for acetylcholine. The Michaelis constants were 72 and 84 mum for acetylcholine and acetylthiocholine, respectively. The cholinesterase was relatively insensitive to eserine (half-maximum inhibition at 0.42 mm) but showed high sensitivity to neostigmine (half-maximum inhibition at 0.6 mum). Other animal cholinesterase inhibitors were also found to inhibit the bean enzyme but most of them at higher concentrations than are generally encountered. Choline stimulated enzymatic activity. The molecular weight of the cholinesterase was estimated to be greater than 200,000, but at least one smaller form was observed. It is suggested that the large form of cholinesterase is converted to the smaller form by proteolysis.     

Some aspects of the control of root growth and georeaction: the involvement of indoleacetic Acid and abscisic Acid

Apical segments of roots of Zea mays L. cv. Orla and cv. Anjou show a strong georeaction during 7 hours geostimulation. This is abolished by detipping the segments and restored by replacing the tips upon the apical cut surfaces. After exodiffusion of endogenous indoleacetic acid (IAA) the retipped segments showed a significantly lower geocurvature. Application of low concentrations of IAA to the basal cut surface of root segments from which endogenous IAA had not been allowed to exodiffuse increased the geocurvature of retipped Orla segments but decreased geocurvature of Anjou segments. At appropriate concentration basally applied IAA restored the georeaction capacity of root segments from which the endogenous auxin had exodiffused. The implications of the interaction between exogenous and endogenous IAA in the control of root georeaction are discussed with special reference to the normal role of endogenous IAA in the regulation of root georeaction and the variation in endogenous IAA content of roots of different cultivars of maize. The probability that the normal control of root growth and georeaction involves concomitant actions in the elongation zone of IAA moving preferentially in the acropetal direction and basipetally transported growth inhibitors (such as abscisic acid) produced in the cap cells is stressed.     

Individual and interactive effects of temperature,carbon dioxide and abscisic acid on mung bean (Vigna radiata) plants

We studied the effects of temperature, carbon dioxide and abscisic acid on mung bean (Vigna radiata). Plants were grown under 26/22°C or 32/28°C (16?h?light/8?h?dark) at 400 or 700?μmol?mol^?1 CO₂ and received ABA application of 0 or 100?μl (10?μg) every other day for three weeks, after eight days of initial growth, in growth chambers. We measured 24 parameters. As individual factors, in 16 cases temperature; in 8 cases CO₂; in 9 cases ABA; and as interactive factors, in 4 cases, each of temperature?×?CO₂, and CO₂?×?ABA; and in 2 cases, temperature?×?ABA were significant. Higher temperatures increased growth, aboveground biomass, growth indices, photochemical quenching (qP) and nitrogen balance index (NBI). Elevated CO₂ increased growth and aboveground biomass. ABA decreased growth, belowground biomass, qP and flavonoids; increased shoot/root mass ratio, chlorophyll and NBI; and had little role in regulating temperature–CO₂ effects.

Abbreviations: A_N: net CO₂ assimilation; E: transpiration; F_v/F_m: maximum quantum yield of PSII; g_s: stomatal conductance; LAR: leaf area ratio; LMA: leaf mass per area; LMR: leaf mass ratio;φPSII: effective quantum yield of PSII; qNP: non-photochemical quenching; qP: photochemical quenching; SRMR: shoot to root mass ratio; WUE: water use efficiency