Identification of 3-(O-beta-Glucosyl)-2-Indolone-3-Acetylaspartic Acid as a New Indole-3-Acetic Acid Metabolite in Vicia Seedlings

A new indole-3-acetic acid metabolite was isolated from broad bean (Vicia faba L. cv Chukyo) seedlings. It was a conjugate of dioxindole-3-acetic acid, aspartic acid, and glucose and was identified as 3-(O-β-glucosyl)-2-indolone-3-acetylaspartic acid (molecular weight 484) from ultraviolet, infrared, nuclear magnetic resonance, and mass spectra. Its natural content in 4-day-old Vicia seedlings was estimated to be 8.6 nanomoles per gram fresh weight. It was suggested that oxidation of indole-3-acetic acid not accompanied by decarboxylation might regulate endogenous level of the hormone.     

Role of auxin and gibberellin in the synthesis of Ascorbic Acid and growth of tissue explants

Coleoptile and root tips ofTriticum aestivum cv. Arnej 624 and those ofAvena sativa cv. Victory (Svalöf) as well as dry excised embryos ofTriticum aestivum cv. Rival (Svalöf) and those ofArachis hypogaea cv. 34 3A. H. were cultivated in media containing various concentrations of sucrose and growth regulators, like ascorbic acid, indole-3-acetic acid and gibberellin. Growth, differentiation and water uptake of the various explants were determined at regular time intervals. Further, the concentration of the endogenous ascorbic acid in mg./g. fresh weight, as well as the amount of this growth regulator utilized as per cent of the total were determined. Although all the three growth regulators promote growth in the explants, their effect is best felt when sucrose of a higher concentration (1.0 per cent) is added to the medium. In fact, the response to 1.0 per cent sucrose is sometimes as good as a combination of a growth regulator with sucrose, especially in the case of root explants. The results clearly indicate that the biosynthesis of ascorbic acid in the explants is catalyzed by the addition of indole-3-acetic acid as well as gibberellin. Simultaneously, the utilization of ascorbic acid is also appreciably increased by the presence of these growth regulators. Addition of 1.0 per cent sucrose to the medium containing the above mentioned growth regulators augments to a considerable extent not only the concentration of ascorbic acid, but also steps up its utilization. Enhancement of ascorbic acid as well as its increased utilization are correlated with rapid imbibition of water, growth and differentiation. The role of ascorbic acid in growth is discussed; and on the basis of the data presented here it is postulated that: (1) auxin and gibberellin function in the growth process by catalyzing the biosynthesis of ascorbic acid; and (2) that ascorbic acid not only participates in activation of various enzyme systems, but also stimulates the production of adenosine triphosphate by acting as an electron donor in photosynthetic phosphorylation as well as oxidative phosphorylation; (3) that the above action of ascorbic acid creates a favourable redox balance for synthesis of nucleic acids, proteins, enzymeproteins, and cell-wall constituents, thus enabling the processes of cell division and enlargement to proceed at a fast rate; and (4) that the relative rates of cell division and cell enlargement as well as “ageing” will determine the pattern of plant development.     

In vitro high frequency plant regeneration from hypocotyl and root segments of spinach by organogenesis

An efficient protocol for spinach (Spinacia oleracea L.) plant regeneration from hypocotyl and root segments was established. When the sub-apical hypocotyl and tip-free root segments were cultured on Murashige & Skoog (1962)-based medium containing high concentrations of indole-3-acetic acid (85.62 M) and gibberellic acid (100 M), more than 75% and 90% of the hypocotyl and root explants, respectively, formed shoots. After elongation, more than 92% of the shoots rooted on medium supplemented with 2.85–5.71 M of indole-3-acetic acid. More than 70% of rooted plantlets survived in soil and were fertile. Significant interactions between growth regulator combinations, explant types and environmental conditions on shoot initiation, development and rooting were discussed.Abbreviations BA benzyladenine - BM Murashige & Skoog basal medium - B5 Gamborg et al. medium (1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - 2ip isopentenyladenine - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige & Skoog medium (1962) - NAA naphthaleneacetic acid - HS hypocotyl segments - RSS root segments of seedlings - RSV foot segments of in vitro plantlets     

Phytohormones,Rhizobium mutants,and nodulation in legumes. IV. Auxin metabolites in pea root nodules

High specific activity [³H]indole-3-acetic acid (IAA) was applied directly to root nodules of intact pea plants. After 24 h, radioactivity was detected in all plant tissues. In nodule and root tissue, only 2–3% of³H remained as IAA, and analysis by thin layer chromatography suggested that indole-3-acetyl-L-aspartic acid (IAAsp) was a major metabolite. The occurrence of IAAsp in pea root and nodule tissue was confirmed unequivocally by gas chromatography-mass spectrometry (GC-MS). The following endogenous indole compounds were also unequivocally identified in pea root nodules by GC-MS: IAA, indole-3-pyruvic acid, indole-3-lactic acid, indole-3-propionic acid, indole-3-butyric acid, and indole-3-carboxylic acid. Evidence of the occurrence of indole-3-methanol was also obtained. With the exception of IAA and indole-3-propionic acid, these compounds have not previously been unequivocally identified in a higher plant tissue.     

The effect of CCC on the levels of endogenous indole-3-acetic acid,abscisic acid,gibberellins and cytokinins inVicia faba plants

Vicia faba plants cv. ‘Erfordia’ were treated with single application of CCC at 250 mg l^?1, 7 days before extraction. Such a concentration resulted in a 10.4, 14, 5 and 3.3 fold, respectively, increase in the levels of endogenous IAA, ABA, gibberellins and cytokinins relative to the controls. The results obtained indicate that a single application of CCC at a low concentration was sufficient to enhance the endogenous growth hormones in the treated plants. The results were obtained using GLC analyses for IAA, ABA and cytokinins, and the lettuce hypocotyl and soybean callus bioassay for gibberellins and cytokinins, respectively.     

Concentrations of Indole-3-acetic Acid and Its Esters in Avena and Zea

An isotope-dilution method has been developed for the assay of free indole-3-acetic acid and ester indole-3-acetic acid as measured by indole-3-acetic acid liberated by mild alkaline hydrolysis. Application of this method to seedlings of Avena sativa and Zea mays indicates the upper limit of free indole-3-acetic acid in Avena to be about 16 μg per kg and in Zea, about 24 μg. The amount of 1 n alkali-labile indole-3-acetic acid in Zea is about 330 μg per kg and there is very little 1 n alkali-labile IAA in Avena. A chemical characterization of the indole-3-acetic acid of Avena and a confirmation of the chemical characterization of the indole-3-acetic acid of Zea is presented.     

Oxidation of Indole-3-acetic Acid and Oxindole-3-acetic Acid to 2,3-Dihydro-7-hydroxy-2-oxo-1H Indole-3-acetic Acid-7′-O-β-d-Glucopyranoside in Zea mays Seedlings

Radiolabeled oxindole-3-acetic acid was metabolized by roots, shoots, and caryopses of dark grown Zea mays seedlings to 2,3-dihydro-7-hydroxy-2-oxo-1H indole-3-acetic acid-7′-O-β-d-glycopyranoside with the simpler name of 7-hydroxyoxindole-3-acetic acid-glucoside. This compound was also formed from labeled indole-3-acetic acid supplied to intact seedlings and root segments. The glucoside of 7-hydroxyoxindole-3-acetic acid was also isolated as an endogenous compound in the caryopses and shoots of 4-day-old seedlings. It accumulates to a level of 4.8 nanomoles per plant in the kernel, more than 10 times the amount of oxindole-3-acetic acid. In the shoot it is present at levels comparable to that of oxindole-3-acetic acid and indole-3-acetic acid (62 picomoles per shoot). We conclude that 7-hydroxyoxindole-3-acetic acid-glucoside is a natural metabolite of indole-3-acetic acid in Z. mays seedlings. From the data presented in this paper and in previous work, we propose the following route as the principal catabolic pathway for indole-3-acetic acid in Zea seedlings: Indole-3-acetic acid → Oxindole-3-acetic acid → 7-Hydroxyoxindole-3-acetic acid → 7-Hydroxyoxindole-3-acetic acid-glucoside.     

Synthesis and Biological Activity of N-Acyl O-Indolylalkyl Ethanolamines

The plant-growth regulators, indole-3-carboxylic acids, were introduced into N-acyl ethanolamines, and a series of N-acyl O-indolylalkyl ethanolamines were prepared. Their biological activities to regulate rape hypocotyl elongation, cucumber cotyledon expansion and common wheat coleoptile growth were tested. The results indicate that the title compounds inhibited rape hypocotyl elongation, especially the indole-3-propionic acid derivatives, whose bioactivity was better than that of indole-3-acetic acid.     

Auxin levels in single half nodes ofAvena fatua estimated using high performance liquid chromatography with coulometric detection

A high performance liquid chromatography (HPLC) based micro-method for estimation of indole-3-acetic acid (IAA) levels in single half nodes from the flowering stalks ofAvena fatua has been developed; this features a dual electrode coulometric electrochemical detector operating at a detection limit of c. 2 pg. Samples were prepared by solvent partitioning and preliminary fractionation with C¹⁸ Sep-Pak cartridges. Two stages of reversed phase ion pair HPLC were employed; the first was gradient elution with fluorescent detection, the second, isocratic elution with coulometric detection. The lower limit for estimation of IAA levels in purified extracts was c. 5 pg.     

Identification of dehydrocostus lactone and 4-hydroxy-β-thujone as auxin polar transport inhibitors

The survey of naturally occurring of auxin polar transport regulators in Asteraceae was investigated using the radish (Raphanus sativus L.) hypocotyl bioassay established in this study. Significant auxin polar transport was observed when radiolabeled indole-3-acetic acid (IAA) was applied at the apical side of radish hypocotyl segments, but not when it was applied at the basal side of the segments. Almost no auxin polar transport was observed in radish hypocotyl segments treated with synthetic auxin polar transport inhibitors of N-(1-naphthyl)phthalamic acid (NPA) and 9-hydroxyfluorene-9-carboxylic acid (HFCA) at 0.5 μg/plant. 2,3,5-Triiodobenzoic acid (TIBA) at 0.5 μg/plant was less effective than NPA and HFCA, and p-chlorophenoxyisobutyric acid (PCIB) at 0.5 μg/plant had almost no effect on auxin polar transport in the radish hypocotyl bioassay. These results strongly suggest that the radish hypocotyl bioassay is suitable for the detection of bioassay-derived auxin polar transport regulators. Using the radish hypocotyl bioassay and physicochemical analyses, dehydrocostus lactone (decahydro-3,6,9-tris-methylene-azulenol(4,5-b)furan-2(3H)-one) and 4-hydroxy-β-thujone (4-hydroxy-4-methyl-1-(1-methylethyl)-bicyclo[3.1.0]hexan-3-one) were successfully identified as auxin polar transport inhibitors from Saussurea costus and Arctium lappa, and Artemisia absinthium, respectively. About 50 and 40 % inhibitions of auxin polar transport in radish hypocotyl segments were observed at 2.5 μg/plant pre-treatment (see “Materials and methods”) of dehydrocostus lactone and 4-hydroxy-β-thujone, respectively. Although the mode of action of these compounds in inhibiting auxin polar transport has not been clear yet, their possible mechanisms are discussed.     

Histogenesis and localization of non-specific esterase in root tip

A procedure was developed for satisfactory freeze-sectioning of root tips. The use of Ca formol-fixed material kept and frozen in Holt's syrup is recommended. The existence and different localization of 2 fractions of non—specific esterase was verified in root tips ofVicia faba. The same results were revealed in fixed and unfixed material. The dynamics ofin situ reaction was followed with respect to optimal incubation time. The results with substrates of different chain length support the existence of 2 fraction of the studied enzyme, none of which, concerning substrate specificity, is a lipase. It follows from the present studies inVicia faba and other species (Cucurbita pepo, Lupinus albus, Pisum sativum Zea mays), that non-specific esterase localization is not directly given by histogenesis.     

Morphogenesis and plant regeneration from tissue cultures of Jatropha curcas

Techniques for the regeneration of Jatropha curcas L. from various explants have been developed. Regeneration from hypocotyl, petiole and leaf explants was evaluated on a range of concentrations of zeatin, kinetin and N⁶ benzyladenine (BA) either singly or in combination with indole-3-butyric acid (IBA). Higher regeneration from hypocotyl and petiole explants was obtained on BA with IBA than on zeatin- or kinetin-supplemented media. Leaf discs from the third expanding leaf exhibited higher regeneration potential than those from the fourth leaf. Independent of the explant type, direct adventitious shoot bud induction was recorded highest on MS medium with 2.22 M BA and 4.9 M IBA. Although the same BA concentration but with reduced IBA concentration (0.49 M) proved effective in callus mediated regeneration from hypocotyl and leaf explants, the petioles required lower concentrations of the two growth regulators (0.44 M BA and 0.49 M IBA). Regenerated shoots could be rooted on growth regulator-free gelled full-strength MS medium. Following simple hardening procedures, the in vitro-raised plants could be transferred to soil and grown to maturity in the field.Abbreviations BA N⁶ benzyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog's (1962) medium - NAA -naphthaleneacetic acid     

Position and extent of the elongation zone in the root tip of the broad beanVicia faba L.

It was found by measuring the length of the cortex cells of the root tips of the broad beanVicia faba L. that the beginning of the elongation zone lies at about 1–2 mm from the initials and its end at about 7–8 mm from the initials. Shrinkage of the object during microtechnical treatment was negligible. The autonomy of the individual tissues of the root tip was taken into account.     

The differentiation of α- and β -glucosidase and α- and β-galactosidase isoenzymes from maize and broad bean root tips using disc electrophoresis in polyacrylamide gels

For the separation of α- and β-glucosidase and α- and β-galactosidase isoenzymes fromZea mays L. andVicia fabaL. root tips the system of disc electrophoresis in polyacrylamide gel developed for basic protein separation proved most suitable. The detection was carried out by a simultaneous azocoupling reaction. In maize α-glucosidase was not detected, β-glucosidase gave 3, α-galactosidase 4, and β-galactosidase 3 zones. In broad bean a- and β-glucosidases were absent, α-galactosidase gave 2 and β-galactosidase 3 zones, α- and β-galactosidase activity zones correspond principially to each other in their position. In maize one zone gives a positive reaction for both β-glucosidase and α- and β-galactosidaso.     

A method to produce encapsulatable units for synthetic seeds in Asparagus officinalis

A method to produce encapsulatable units for synthetic seeds was developed in Asparagus officinalis L. Encapsulatable units with high conversion ability in non-sterile soil were produced from somatic embryos by a pre-encapsulation culture. The synthetic seeds containing somatic embryos without the pre-encapsulation culture did not germinate in soil. When the pre-encapsulation culture medium did not contain growth regulators, the roots elongated too much to accomplish encapsulation. Several growth regulators were studied and indole-3-acetic acid was considered to be optimum at 28.5 M. The pre-encapsulation culture medium with indole-3-acetic acid inhibited the growth of roots during the pre-encapsulation culture and produced compact encapsulatable units. The growth of roots was promoted when plants were produced from the encapsulatable units. The percent conversion of the synthetic seeds with these encapsulatable units was 72% in non-sterile soil. This is the first report on synthetic seeds in Asparagus officinalis L.     

Inactivity of Oxidation Products of Indole-3-acetic Acid on Ethylene Production in Mung Bean Hypocotyls

The suggestion that indole-3-acetic acid (IAA)-stimulated ethylene production is associated with oxidative degradation of IAA and is mediated by 3-methyleneoxindole (MOI) has been tested in mung bean (Phaseolus aureus Roxb.) hypocotyl segments. While IAA actively stimulated ethylene production, MOI and indole-3-aldehyde, the major products of IAA oxidation, were inactive. Tissues treated with a mixture of intermediates of IAA oxidation, obtained from a 1-hour incubation of IAA with peroxidase, failed to stimulate ethylene production. Furthermore, chlorogenic acid and p-coumaric acid, which are known to interfere with the enzymic oxidation of IAA to MOI, had no effect on IAA-stimulated ethylene production. Other oxidation products of IAA, including oxindole-3-acetic acid, indole-3-carboxylic acid, (2-sulfoindole)-3-acetic acid, and dioxindole-3-acetic acid, were all inactive. 1-Naphthaleneacetic acid was as active as IAA in stimulating ethylene production but was decarboxylated at a much lower rate than IAA, suggesting that oxidative decarboxylation of auxins is not linked to ethylene production. These results demonstrate that IAA-stimulated ethylene production in mung bean hypocotyl tissue is not mediated by MOI or other associated oxidative products of IAA.     

Regeneration of Picea omorika plants via organogenesis

Picea omorika plants were regenerated from embryo and seedling shoot tip cultures. Adventitious and axillary shoots were produced on 1/2 MS medium containing benzyladenine and kinetin. Benzyladenine was more effective in bud induction, whereas kinetin hastened shoot development. Excised shoots were elongated on 1/3 MS medium without growth regulators, multiplied with kinetin and rooted with or without indole-3-butyric acid.Abbreviations BA N⁶-benzyladenine - 2IP N ⁶-(²-isopenteny) adenine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

The localization and the isoenzymes of α- and β-Galactosidases in root tips

The localization was studied of α- and β-galactosidases in frozen sections of Ca-formol fixed root tips using simultaneous azocoupling reaction. In all species studied (Allium cepa,Cucurbita maxima, Lupinus albus, Pisum sativum, Vicia faba, Zea mays) positive results were obtained, the localization being ubiquitous (according to localization typology given here). InVicia faba andZea mays the isoenzymes of α- and β-galactosidases were revealed by means of acrylamide gel electrophoresis, using authors’ modification of Reisfeld method, in whole root tips, particular growth zones and separately in cortex and central cylinder. No differences were observed comparing stele and cortex. Whereas characteristic isoenzyme patterns were found in individual growth zones in maize, no differences appeared in broad bean. A comparison was made of thein situ localization and of the isoenzyme patterns of α- and β-galactosidases with α- and β-glucosidases. In the case of galactosidases, positive results appear with both α- and β-galactoside. The rising of pH to neutrality leads to considerable decrease in the activity of both galactosidases.     

In vitro micropropagation of Basilicum polystachyon (L.) Moench and identification of endogenous auxin through HPLC

An efficient plant regeneration protocol was developed for Basilicum polystachyon (L.) Moench using shoot tip from in vitro germinated plant. Both shoot multiplication and root induction were initiated from shoot tip explants in Murashige and Skoog’s (MS) basal medium supplemented with N⁶-benzylaminopurine (BAP) and 6-furfurylaminopurine (Kin) combination with 1-naphthaleneacetic acid (NAA) and without any plant growth regulator. Among the different concentrations and combinations of growth regulators, the highest number of shoots per explants was induced on 13.32 μM BAP with 0.53 μM NAA. It was also found that the multiplication of shoots along with roots induced in MS medium without any plant growth regulators. The in vitro grown plants were successfully hardened and acclimatized in the field with a 99% survival rate. The results obtained from HPLC analysis established the presence of a significant amount of endogenous auxin, viz. indole-3-acetic acid acid and indole-3-butyric acid in the shoot and root tips of B. polystachyon. This is the first report of a successful multiplication of B. polystachyon in absence of plant growth regulators and the presence of an abundant quantity of endogenous auxin in root and shoot tips using Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) coupled with ultraviolet–visible (UV–Vis) detector.