Transport and accumulation of indole-3-acetic acid in pea cuttings under two levels of irradiance

The transport and accumulation of 2-[¹⁴C]-IAA applied to the apex of cuttings of Pisum sativum L. cv. Alaska was greater in cuttings from stock plants grown under 38 W m⁻² than 16 W m⁻². Accumulation of ^14C in the base of the cuttings from the highest level of irradiance was correspondingly more significant. The level of irradiance to the stock plants greatly affected the rate of accumulation, while the light conditions during IAA transport had a minor effect. The amount of IAA reaching the base of the cuttings increased with increasing concentration of IAA in the treatment solution, but the percentage of applied IAA reaching the base decreased.
The relative chromatographic partition of ethanol-extractable ¹⁴C showed that, after 12 h of IAA-transport, the amount of 2-[¹⁴C]-IAA was higher in the base of cuttings from 38 W m⁻² than in those from 16 W m⁻². After a further 12 h of transport the relative amounts of 2-[¹⁴C]-IAA in the two types of cuttings were reduced to the same lower level.
A possible role of an irradiance-mediated difference in the topographic distribution of IAA in the base of pea cuttings on the subsequent adventitious root formation is discussed.     

Metabolism of [14C]-indole-3-acetic acid by soybean callus and hypocotyl sections

Metabolism of indole-3-acetic acid in soybean [ Glycine max (L.) Merr.] was investigated with [1-¹⁴C]- and [2-¹⁴C]-indole-3-acetic acid (IAA) applied by injection into soybean hypocotyl sections and by incubation with soybean callus. Free IAA and its metabolites were extracted with 80% methanol and separated by high performance liquid chromatography with [³H]-IAA as an internal standard. Metabolism of IAA in soybean callus was much greater than that in tobacco ( Nicotiana tabacum L.) callus used for comparison. High performance liquid chromatography of soybean extracts showed at least 10 metabolite peaks including both decarboxylated and undecarboxylated products. A major unstable decarboxylated metabolite was purified. [¹⁴C]-indole-3-methanol (IM) was three times more efficient than [2-¹⁴C]-IAA as substrate for producing this metabolite. It was hydrolyzable by β-glucosidase (EC 3.2.1.21), yielding an indole and D-glucose. The indole possessed characteristics of authentic IM. Thus, the metabolite is tentatively identified as indole-3-methanol-β-D-glucopyranoside. The results suggest that soybean tissues are capable of oxidizing IAA via the decarboxylative pathway with indole-3-methanol-glucoside as a major product. The high rate of metabolism of IAA may be related to the observed growth of soybean callus with high concentrations of IAA in the culture medium.     

Catabolism of indole-3-acetic acid to indole-3-methanol in a crude enzyme extract and in protoplasts from Scots pine (Pinus sylvestris)

The antagonistic effects of ethylene and Ag⁺ on the metabolism of [1-¹⁴C]indole-3-acetic acid (IAA) and on the rates of ethylene production were studied in tobacco leaf discs ( Nicotiana rustica var. Brasilia ). During the first 10 h of incubation, Ag⁺-pretreated leaf discs contained more free [¹⁴C]IAA than untreated ones due to decreased oxidative decarboxylation, and the discs also produced more ethylene. Exogenously supplied ethylene nullified these effects of Ag⁺. However, the most pronounced effect of Ag⁺ in increasing ethylene production, as well as the strongest antagonistic effect of exogenous ethylene, were found between 24 and 48 h of incubation. During this time span no effect on the level of free IAA and on its decarboxylation could be observed. It is suggested that ethylene exerted its autoinhibitory effect by a feedback control on the IAA-induced ethylene biosynthesis. Possible mechanisms for the autoinhibitory effect of ethylene are discussed.     

Indole-3-methylglucosinolate biosynthesis and metabolism in clubroot diseased plants

lndole-3-methylglucosinolate biosynthesis and metabolism in roots of Brassica napus (swede, cv. Danestone II) infected with Plasmodiophora brassicae Wor. were investigated with a pulse feeding technique developed to infiltrate intact tissue segments with labelled substrates. Infected root tissue metabolized [¹⁴C]-L-tryptophan to indole-3-methylglucosinolate, indole-3-acetonitrile, and some other lipophilic indole compounds. The incorporation of radioactivity into these compounds was significantly enhanced in infected tissue compared with control tissue. A time course study showed a high turnover of indole-3-methylglucosinolate and indole-3-acetonitrile in infected tissue. However, thioglucoside glucohydrolase activity was not changed in infected tissue compared with control tissue. Disc electrophoresis revealed the same isoenzyme in both tissues. Control and infected tissues both rapidly hydrolyzed [¹⁴C]-indole-3-acetonitrile in vivo. The possibility of a disease specific biosynthesis of indole-3-acetic acid from indole-3-methylglucosinolate as the result of a changed compartmentation is discussed.     

l-Tryptophan metabolism in wound-activated and Agrobacterium tumefaciens-transformed potato tuber cells

The in vivo metabolism of L-tryptophan in wound-activated and Agrobacterium tumefaciens , strain C 58, transformed tissues of white potato tubers ( Solanum tuberosum L. cv. Saskia) was investigated. The following metabolites of L-tryptophan were identified in both tissues by co-chromatography with authentic standards in several thinlayer chromotography (TLC) and high pressure liquid chromatographic (HPLC) systems: indole-3-acetic acid (IAA), indole-3-acetaldehyde, indole-3-ethanol, indole-3-acetamide and tryptamine. Labelled indole-3-acetaldoxime was only found in transformed tissue. Crown gall tissue generally incorporated [¹⁴C]-L-tryptophan into precursors of IAA at a distinctly higher rate than did wound tissue. Tryptamine and indole-3-ethanol accumulated about ten-fold more label in crown gall cells than in cells from wounded tissue. The incorporation of radioactivity into indole-3-acetamide as determined by 2 consecutive TLC systems followed by HPLC analysis was rather low, though consistently observed in both tissues. An indole-3-acetamide hydrolyzing enzyme, the putative product of gene 2 on the T-DNA, could be extracted from the transformed tissue only. The indole-3-ethanol level was 4.3 nmol (g dry weight)⁻¹ and 41 nmol (g dry weight)⁻¹ for wounded tissue and primary crown gall tissue, respectively, as determined by HPLC with a [¹⁴C]-labelled internal standard. The experiments are critically discussed in relation to recent reports on a T-DNA encoded enzyme of IAA biosynthesis in crown gall tumors.     

Time-course of uptake of dissolved inorganic carbon through willow roots in light and in darkness

Uptake of dissolved inorganic carbon (DIC) from a nutrient solution by willow roots was measured in light and darkness and the distribution in the plant of DIC taken up by the roots was determined. It was also studied whether the transport system could be activated by preincubation with dissolved inorganic carbon.
Willow plants ( Salix cv. Aquatica gigantea) grown in hydroponic culture media were preincubated for 2 days with or without 0.74 mM NaHCO₃. After preincubation, either unlabelled or [¹⁴C]-labelled NaHCO₃ was injected into the media and after 1, 5, 10 and 24 h either in light or in darkness the plants were harvested in pieces into liquid nitrogen, lyophilized and burned in a combustion chamber.
¹⁴C was transported through the roots to the shoots and leaves both in light and in darkness, although incorporation of ¹⁴C in darkness was only half of that in light at the end of the 24-h feeding period. Both in light and in darkness the amount of ¹⁴C increased in all parts of willow plants with time. In light the rate of labelling was highest into cuttings and shoots. In darkness more than half of the total label was detected in cuttings of both the non-activated and the activated treatments.
In the shoots the middle part was most strongly labelled after 5 and 10 h, but after 24 h ¹⁴C moved towards the base of the shoot. In the leaves at all feeding times most radioactivity was incorporated into the young, fully open leaves on the upper part of the shoots. Preincubation of plants with unlabelled NaHCO³ in growth media had no clear effect on the rate of DIC uptake either in light or in darkness.     

Distribution and metabolism of root-applied cytokinins in Pisum sativum

When N ⁶ [8–¹⁴C] furfuryladenine was applied to the intact root system of Pisum sativum L. cv. Meteor seedlings it was almost completely metabolised to other compounds within 24 h. Of the total activity recovered from the plants 94.5% was retained in the root system itself. ¹⁴C was recovered in a number of ethanol-soluble compounds and in ribonucleic acid, deoxyribonucleic acid and protein fractions of roots, stems, leaves and axillary buds. In rapidly growing axillary buds released from apical dominance by removal of the shoot apex the combined nucleic acid fractions accounted for 63.3% of the total ¹⁴C recovered from these organs. Xylem exudate collected from decapitated plants 0 to 12 h after supplying N ⁵[8–¹⁴C]furfuryladenine to the roots consistently contained a single major ¹⁴C-labelled compound which, in three different solvent systems, had the same Rf values as a major endogenous cytokinin isolated from the xylem of unlabelled plants. The content of N ⁶ [8–¹⁴C] furfuryladenine itself in the xylem exudate was always low and in some experiments it could not be detected.
It is suggested that part of the label from N ⁶ [8- ¹⁴CJfurfuryladenine taken up by the intact root system may have become incorporated in an endogenous cylokinin before export to the shoot.     

Conversion of indole-3-acetaldoxime to indole-3-acetonitrile by plasma membranes from Chinese cabbage

The in vitro conversion of [¹⁴C]-indole-3-acetaldoxime (IAOX) to [¹⁴C]-indole-3-acetonitrile (IAN) by plasma membranes enriched by aqueous two-phase partitioning of Chinese cabbage ( Brassica campestris L. ssp. pekinensis cv. Granat) has been studied. The reaction product was identified by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC). A reducing agent, e.g. ascorbic acid, was needed as cofactor for the formation of IAN from IAOX. Reduction equivalents and metal ions were not involved in the conversion of IAOX to IAN. The pH optimum for the reaction was at 6.0 and the apparent K_m for IAOX was 6.3 μ M . The enzyme was not inhibited by thiol reagents. The pI of the enzyme was determined to be 7.1 by isoelectric focusing (IEF). Gel permeation chromatography showed one major activity peak of 40 kDa. The reaction is considered as part of a channeling process leading from tryptophan to IAN with IAOX as an intermediate. This process is probably regulated by the indole derivatives IAOX and IAN.     

Regulation of cell wall synthesis by auxin and fusicoccin in different tissues of pea stem segments

Cell wall synthesis was studied by determining the incorporation of [¹⁴C]-glucose into epidermal and cortical cell walls of etiolated Pisum sativum L. cv. Alaska stem segments. Walls were fractionated into the matrix and cellulose components, and incorporation into these components assessed in terms of the total uptake of label into that tissue. When segments were allowed to elongate, the stimulation of total glucose uptake by indole-3-acetic acid (IAA) and fusicoccin (FC) was greater than their stimulation of incorporation. IAA and FC thus did not stimulate precursor incorporation in elongating segments. When elongation was inhibited by calcium, however, IAA and FC significantly promoted wall synthesis in the cortex and vasular tissue (which shows almost no growth or acidification response to auxin). In these tissues incorporation into matrix and cellulose was promoted approximately equally. In the epidermis (thought to be the tissue responsive to auxin in the control of growth), FC promoted a significant increase in wall synthesis, although less than that in the cortex, while there was some evidence of a similar promotion by IAA. Both IAA and FC had a greater effect on incorporation into the matrix component of the wall than into cellulose. The results that FC caused a substantial promotion of cell wall synthesis which was not due solely to elongation, and that the inner non-growth responsive cortical tissues can respond to IAA. Moreover, a comparison of the effects of IAA and FC on the different components of the wall suggests that the response in the epidermis differs from that in the other tissues.     

Analysis of [14C] indole-3-acetic acid metabolites from the primary roots of Zea mays seedlings using reverse-phase high-performance liquid chromatography

Methanolic extracts of Zea mays L. cv. Fronica root segments which had been incubated in [¹⁴C] indole-3-acetie acid were analysed by reverse-phase high-performance liquid chromatography. Metabolism of indole-3-acetic acid was found to be rapid and extensive with at least 11 products apparent after a 2 h incubation. A comparison of metabolites of [1-¹⁴C]– and [2-¹⁴C] IAA, calculations of ¹⁴CO₂ evolution, and data on the polarity of products indicated that decarboxylation had not occurred. An average of 34% of the radioactivity remained associated with the indole-3-acetic acid peak.     

Allocation of carbon to growth, storage and respiration in the vegetative barley plant

Abstract. It is proposed that the growing plant can be divided into three compartments with reference to carbon: soluble, storage and structural. Experiments carried out at 10, 15, 20 and 30°C in the light followed changes in size of these compartments in barley plants 10–24 days old. The redistribution of ^I4C photo-assimilated by 10 day old plants was monitored simultaneously. The soluble and storage compartments are a higher percentage of plant weight at lower temperatures, and are turned over rapidly at all temperatures; they form the source of respired ¹⁴C. About 30% of the ¹⁴C fixed enters structural material; in the first 24 h after labelling, for each unit of ¹⁴C entering the structural compartment, between 0–9 (at 15°C) and 3.2 (at 30°C) units of ¹⁴C are lost by respiration. At 15°C in the dark, respiratory loss of ¹⁴C is initially from soluble and storage compartments; thereafter respiration of ^I4C occurs at the expense of structural material.     

ACETYLCHOLINE METABOLISM AND CHOLINE UPTAKE IN CORTICAL SLICES

Abstract— The uptake of [¹⁴C]choline was studied in cortical slices from rat brain after their incubation in a Krebs-Henseleit medium containing either 4.7 m m -KCl (low K), 25 m m -KCl (high K) or 25 m m -KCl without calcium (Ca free, high K). With 0.84 μ m -[¹⁴C]choline in the medium the uptake per gram of tissue was 0.62 nmol after incubation in low K medium, 1.13 nmol after incubation in high K medium and 0.78 nmol after incubation in a Ca free, high K medium. The differences caused by potassium were greater in fraction P₂ than in fractions P₁ and S₂. With 17 and 50 μ m -[¹⁴C]choline in the medium greater amounts of [¹⁴C]choline were taken up, but the effect of potassium on the uptake almost disappeared. The amount of radioactive material in fraction P₂ followed Michaelis-Menten kinetics with K _m values of 2.1 and 2.3 μ m after incubation in low and high K medium, respectively. Hemicholinium-3 only slightly inhibited choline uptake from a medium with 0.84 μ m -[¹⁴C]choline, but it abolished the extra-uptake induced by high K medium. The radioactivity in the slices consisted mainly of unchanged choline and little ACh was formed after incubation in low K medium, but after incubation in high K medium 50% of the choline taken up was converted into ACh. The hemicholinium-3 sensitive uptake of choline, the conversion of choline into ACh and the synthesis of total ACh, were stimulated about 7–8-fold by potassium. It is concluded that in cortical slices from rat brain all choline used for the synthesis of ACh is supplied by the high-affinity uptake system, of which the activity is geared to the rate of ACh synthesis.     

Localization of newly synthesized indoIe-3-methylglucosinolate (= glucobrassicin) in vacuoles from horseradish (Armoracia rusticana)

The presence and concentration of indole-3-methylglucosinolate [= glucobrassicin; 0.49 μmol (g dry weight)-1] and its 1-methoxy derivative [0.38 umol (g dry weight)⁻¹] in Armoracia rusticana P. Gärtner, B. Meyer and Scherb, storage roots were measured. The storage tissue contains L-tryptophan [1.63 μmol (g dry weight)^-1], which is the common precursor amino acid of the indoleglucosinolates. Tissue cylinders convert [¹⁴C]- l -tryptophan efficiently to the indoleglucosinolates (25%) in vivo. The conversion of [¹⁴C]- l -tryptophan to indole-3-methylgluco-sinolate shows biphasic kinetics.
A fraction rich in vacuoles was prepared from tissue sections to which [¹⁴C]- l -tryp-tophan had been fed and allowed to be metabolized. The predominantly vacuolar location of both L-tryptophan and the indoleglucosinolates is demonstrated by correlation with the vacuolar marker acid phosphatase.
The significance for the regulation of the indoleglucosinolate biosynthesis and the role of indole-3-methylglucosinolate as a potential auxin precursor are discussed.     

The effect of water status and season on the incorporation of 14CO2 and [14C]-acetate into resin and rubber fractions in guayule

The effects of drought stress and season on both allocation of photosynthates to stems and leaves and potential for stem rubber synthesis were studied in guayule ( Parthenium argentatum Gray USDA line 11604). Two-year-old plants grown under field conditions in the Negev desert of Israel were subjected to different irrigation regimes, and water status was assessed by measuring the relative water content (RWC). Undetached plant tips were exposed to a 1 h pulse of ¹⁴CO₂, followed by a 24 h chase. ¹⁴C fixed and translocated to different plants parts and notably ¹⁴C incorporation into rubber and resin fractions was determined. The potential of detached branch slices to incorporate [¹⁴C]-acetate into rubber was also studied. A higher percentage of fixed ¹⁴C was translocated from shoot tips in winter (28–30%) than in summer (15–18%). The percentage of [¹⁴C]-acctate incorporated into the rubber fraction by stem slices was maximal in winter (20%) and minimal in summer (3–5%) in both cases in the absence of drought stress. In summer the translocation of photosynthates into stems was inversely related to plant RWC, dropping from 18% three days after irrigation to 3% 14 days later, and the potential of stems to synthesise rubber was high under drought conditions and low in well irrigated plants.     

Lumiflavin and Lumichrome Transport in the Central Nervous System

Abstract: The transport of the lipid-soluble sugarless flavins, [¹⁴C]lumiflavin and [¹⁴C]lumichrome, into and from the isolated choroid plexus and brain slices was studied in vitro. The isolated choroid plexus accumulated both [¹⁴C] flavins by a saturable, energy-requiring process that did not depend on binding or intracellular metabolism of the [¹⁴C] flavins. Both sugar-containing and sugarless flavins, as well as cyclic organic acids, significantly inhibited [¹⁴C]lumiflavin and [¹⁴C]Iumichrome uptake by the isolated choroid plexus. Within 2.5 min, 75% of the [¹⁴C]lumiflavin accumulated by the isolated choroid plexus was released into the medium. Brain slices accumulated [¹⁴C]lumiflavin by a saturable process that did not meet all the criteria for active transport. Ninety-five percent of the [¹⁴C]lumiflavin accumulated by brain slices was released into the medium within 7.5 min. In vivo , 2 h after the intraventricular injection of 6.5 nmol [¹⁴C]lumiflavin, almost all of the [¹⁴C]flavin was cleared from the CNS. Addition of 3.5 μmol FMN to the intraventricular injectate significantly decreased the clearance of [¹⁴C]lumiflavin from the CNS. These studies document that the sugarless flavins are transported by the flavin transport systems in the CNS.     

SYNTHESIS AND RELEASE OF [14C]ACETYLCH0LINE IN SYNAPTOSOMES

Abstract— Synaptosomes took up [¹⁴C]choline, about half or more of which was converted to [^I4C]acetylcholine when incubated in an appropriate medium containing 1 to 5 μ M-[¹⁴C] choline and neostigmine. The amount of [¹⁴C]acetylcholine synthesized in synaptosomes increased in parallel with the increase of Na⁺ concentration in the incubation medium. The effect of Na⁺ on the uptake of [^I4C]choline into synaptosomes was dependent on the concentration of choline in the incubation medium.
About 25 per cent of [¹⁴C]acetylcholine synthesized in synaptosomes was released rapidly into the medium by increasing the K⁺ concentration in the medium from 5 m m to 35 m m . The change of Na⁺ concentration hardly affected the release of [¹⁴C]acetylcholine. The effect of K⁺ on the release of [¹⁴C]choline was rather small compared to that on [¹⁴C] acetylcholine. Ouabain promoted the release of [¹⁴C]acetylcholine.     

Effect of zeatin on the growth and indolyl-3-acetic acid and abscisic acid levels in maize roots

Elongation, indolyl-3-acetic acid (IAA) and abscisic acid (ABA) levels, – gas chromatography-mass spectrometry quantification –, in the elongating zone were analysed for maize ( Zea mays L., Cv. LG11) roots immersed in buffer solution with or without zeatin (Z). The effect of Z depends on the initial extension rate of roots. The slower growing roots are more strongly inhibited by Z (10⁻⁷−10₋₅ M ) and they show a greater increase in IAA and ABA content. When compared to the rapidly growing roots, the larger reactivity of the 'slow'ones cannot be attributed to a higher Z uptake as shown when using [¹⁴C]-Z. It is suggested that Z could regulate root elongation by acting on the IAA and/or ABA level. The comparative action of these two hormones is discussed.     

Alanyl turnover from lipoteichoic acid to teichoic acid in Staphylococcus aureus

Abstract The metabolism of d -alanyl substituents of lipoteichoic acid (LTA) and teichoic acid was studied in Staphylococcus aureus . Double labelling with [³H]glycerol and d -[¹⁴C]alanine revealed that during the chase LTA was stable whereas its ¹⁴C label rapidly decreased. Half-time comparison indicated an enzyme- rather than a base-catalyzed process. Correlated with the loss of [¹⁴C]alanine from LTA was an increase of the radioactivity in wall-linked alanine ester which, after hydrolysis with HF, proved to be linked to teichoic acid. These results suggest that LTA-alanine is the donor for alanine esterification of teichoic acid. In connection with previous data we hypothesize that the loss of alanine from LTA is compensated by de novo incorporation.     

Origin and dynamics of indoleacetic acid under polar transport in Pinus sylvestris

Polarly‐transported IAA is regarded as a long distance correlative signal important in many aspects of plant physiology, including, for example, apical dominance and growth and development of vascular tissues. In this study, we investigated the importance of apical sources in supplying stem tissues with IAA. The current‐yearshoots of 4‐year‐old Pinus sylvestris L. saplings were replaced with a source of [¹³C₆]IAA. Subsequent mass spectrometric analysis showed that most of the IAA present at two positions in the subjacent 1‐year‐old internode consisted of [¹³C₆]IAA, while [¹²C]IAA of endogenous origin formed a minor pool. However, the pool of [¹³C₆]IAA decreased from 90 to 80% of the total free IAA pool [¹³C₆]IAA+[¹²C]IAA) while being transported down the shoot. This dilution with [¹²C]IAA indicates that de novo biosynthesis of IAA occurred. An additional defoliation experiment showed that the synthesis took place in stem tissues rather than in the mature leaves. The results confirm the role of apical shoots as the major source of polarly‐transported IAA, but also indicate that synthesis of IAA takes place in stem tissues. This is important when considering IAA balance at the whole plant level.