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.     

Structural analysis of the nit2/nit1/nit3 gene cluster encoding nitrilases,enzymes catalyzing the terminal activation step in indole-acetic acid biosynthesis in Arabidopsis thaliana

A 13.8 kb DNA sequence containing the promoters and the structural genes of the Arabidopsis thaliana nit2/nit1/nit3 gene cluster has been isolated and characterized. The coding regions of nit2, nit1 and nit3 spanned 1.9, 1.8 and 2.1 kb, respectively. The architecture of the three genes is highly conserved. Each isoform consists of five exons separated by four introns. The introns are very similar with respect to size and position, but differ considerably in sequence composition. In contrast to the coding sequences the three promoters are very different in sequence, size and in their repertoire of cis elements, suggesting differential regulation of the three nitrilase isoenzymes by the developmental program of the plant and by diverse environmental factors. The nit1 promoter was subjected to analysis in planta. Translational fusions placing the nit1 full-length promoter and a series of 5-deletion fragments in front of the uidA gene encoding -glucuronidase (GUS) were used for Agrobacterium tumefaciens-mediated transformation of Nicotiana tabacum. GUS expression was highest in fully expanded leaves and in the shoot apex as well as in the apices of developing lateral buds, whereas the GUS activity displayed by developing younger leaflets was restricted to the tips of the expanding leaves. Within the root tissue GUS expression was restricted to the root tips and the tips of newly forming lateral roots. Structural features of the nitrilase gene family and nitrilase gene expression patterns are discussed in context with current knowledge of auxin biosynthesis and auxin effects on different tissues.     

Chlorophyll-protein levels and degree of thylakoid stacking in radish chloroplasts from high-light, low-light and bentazon-treated plants

Lemna gibba plants were incubated aseptically on medium containing labelled 10^-7 M indole-3-acetic acid (IAA-1-¹⁴C). Most of the radioactivity disappeared from the culture medium during a 24 h light period. A high percentage of the loss was due to photolysis and only a low percentage of the radioactivity was recovered in the plants. Uptake of ¹⁴C by the plants was strongly stimulated by light. The radioactivity taken up by the plants was the sum of photosynthetically taken up ¹⁴CO₂ and ¹⁴C taken up in IAA. Analyses with the indolo-α-pyrone fluorescence method revealed that the free IAA content was almost the same in plants grown in control and in IAA media for 5 h, whereas the amount of IAA which could be liberated by alkaline hydrolysis was doubled by the presence of IAA in the medium.     

Influence of extraction solvent (buffer, methanol, acetone) and time on the quantification of indoIe-3-acetic acid in plants

The effect of extraction solvent and time on the measured indole-3-acetic acid (IAA) level was investigated in plant materials having different contents of lAA-conjugates, Tissues from pine ( Pinus sylvestris L.). tobacco ( Nicotiana tabacum L.), and maize ( Zea mays L.) were extracted for 1–9 h with Na-phosphate buffer (pH 7.5). 80% methanol and 70% acetone. IAA was measured by combined gas chromatography-selected ion minitoring-mass spectromctry (GC-SIM-MS) with [¹³C₆]-IAA as an internal standard.
Extraction of maize seedlings with buffer gave a higher estimate of free IAA than did extraction with methanol or acetone, which produced similar values. The increase in free IAA after buffer extraction was paralleled by a stoichiometric decrease in lAA-ester conjugates, indicating that free IAA was formed during buffer extraction by hydrolysis of these conjugates, which are abundant in maize seedlings. The amount of hydrolysis during a 1-h extraction period was estimated to be ca 3% of the total lAA-ester pool. However, in the pine extraxylary tissues and tobacco in-ternodes which lack a significant lAA-ester pool, buffer extraction resulted in the same IAA estimate as extraction with the organic solvents, but produced a cleaner extract. For all the plant materials investigated, a 1-h extraction period was sufficient for equilibrating the internal standard with the endogenous IAA pool.     

Action of Rice α-Glucosidase on Maltose and Starch

Rice seeds possess α-glucosidase I and II, and the action of the α-glucosidases on maltose and starch was studied. The activity on starch was increased 2.3~2.6 times in both enzymes at the concentration of 50 mM of potassium chloride. Such activation was also caused by mono and di-valent cations. The activity on maltose was not influenced by the cations. In mixed substrate experiments, liberation of ¹⁴C-glucose from ¹⁴C-maltose was not inhibited in the presence of starch, and this was also the case with that from ¹⁴C-starch in the existence of maltose. From these results, it was suggested that the α-glucosidases possess maltose-hydrolyzing site and starch-hydrolyzing site separately, and also probably regulatory. The α-glucosidases liberated only glucose from starch, and were presumed to complete hydrolysis of starch after longer incubation.     

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.     

Development of flower buds in thin-layer cultures of floral stalk tissue from tobacco: Role of hormones in different stages

The in vitro development of flower buds was studied on tissue explants of epidermis and subepidermal cortex from the flower stalks of Nicotiana tabacum L. cv. Samsun. The number of flower buds formed depended mainly on cytokinin concentration. Auxin acted as a modifier in a complex way. In early development, NAA at 1 μ M decreased the number of buds initiated and delayed bud emergence. At a later stage, auxin promoted bud outgrowth at the same concentration. Optimal results were obtained when explants were first incubated at low auxin concentration for 3–5 days and subsequently transferred to an elevated auxin level. Physiological processes that lead to flower bud initiation start very soon after the onset of incubation. This was inferred from experiments whereby explants were first cultured at an inductive cytokinin concentration and then transferred to a non-inductive hormone level.     

Effect of 2,5-norbornadiene on abscission and ethylene production in citrus leaf explants

Citrus ( Citrus sinensis L. Osbeck) leaf explants completely abscise within 48 h when exposed to saturating amounts of ethylene at 25°C. When 2,5-norbornadiene was added, 2000 μl 1⁻¹ reduced abscission of explants also exposed to 2 μl 1⁻¹ of ethylene to the level of the control, and 8000 μl 1⁻¹ reduced abscission in explants exposed to 10 μl 1⁻¹ of ethylene to the level of the control, but abscission was complete when 1 000 μl 1⁻¹ of ethylene was used in the presence of 8 000 μl 1⁻¹ of 2,5-norbornadiene. When explants were exposed to 2 μl 1⁻¹ of ethylene, 2000 μl 1⁻¹ of 2,5-norbornadiene prevented abscission if applied up to 10 h after exposure to ethylene. After 18 h, applied 2,5-norbornadiene had little effect on abscission at 48 h. A Lineweaver-Burk plot gave a 1/2 maximum value of 0.12 μl 1⁻¹ for ethylene on abscission, 2,5-Norbornadiene gave competitive kinetics with respect to ethylene with a K₁ value of approximately 120 μl 1⁻¹ of 2,5-norbornadiene. The presence of 2,5norbornadiene stimulated ethylene production, which progressively increased as the 2,5-norbornadiene concentration was increased from 250 to 8 000 μl 1⁻¹ 2,5-Norbornadiene also suppressed the induction of cellulase and polygalacturonase by ethylene. Together, 2,5-norbornadiene and 2,4-dichlorophenoxyacetic acid were more effective than either alone in reducing abscission. 2,5-Norbornadiene also was effective in preventing the reduction of indole-3-acetic acid transport induced by ethylene.     

An NMR method for the study of proton transport across phospholipid vesicles

We have identified [1-¹⁴C]-oxindole-3-acetic acid as a catabolic product of [1-¹⁴C]-indole-3-acetic acid metabolism in Zea mays seedlings. The isolation, and chemical and mass spectral characterization of oxindole-3-acetic acid from corn kernel tissue is described together with data suggesting oxindole-3-acetic acid to be a major catabolic product of indole-3-acetic acid.     

Promotion of stem elongation by indole-3-butyric acid in intact plants of Pisum sativum L.

While indole-3-butyric acid (IBA) has been confirmed to be an endogenous form of auxin in peas, and may occur in the shoot tip in a level higher than that of indole-3-acetic acid (IAA), the physiological significance of IBA in plants remains unclear. Recent evidence suggests that endogenous IAA may play an important role in controlling stem elongation in peas. To analyze the potential contribution of IBA to stem growth we determined the effectiveness of exogenous IBA in stimulating stem elongation in intact light-grown pea seedlings. Aqueous IBA, directly applied to the growing internodes via a cotton wick, was found to be nearly as effective as IAA in inducing stem elongation, even though the action of IBA appeared to be slower than that of IAA. Apically applied IBA was able to stimulate elongation of the subtending internodes, indicating that IBA is transported downwards in the stem tissue. The profiles of growth kinetics and distribution suggest that the basipetal transport of IBA in the intact plant stem is slower than that of IAA. Following withdrawal of an application, the residual effect of IBA in growth stimulation was markedly stronger than that of IAA, which may support the notion that IBA conjugates can be a better source of free auxin through hydrolysis than IAA conjugates. It is suggested that IBA may serve as a physiologically active form of auxin in contributing to stem elongation in intact plants.     

Analysis of microspore-specific promoters in transgenic tobacco

In order to modify the early stages of pollen development in a transgenic context microspore-specific promoters are required. We tested two putatively microspore-specific promoters, the Bp4 promoter from rapeseed and the NTM19 promoter from tobacco. Expression of the gus and barnase reporter genes under the control of these two promoters was studied in transgenic tobacco. Contrary to expectations, the Bp4 promoter became active only after the first pollen mitosis, and not in the microspores. The NTM19 promoter turned out to be highly microspore-specific and directed very high levels of gus expression to the unicellular microspores. The NTM19-barnase transgene caused cell-autonomous death at the mid-unicellular microspore stage, whereas Bp4-barnase induced cell ablation of early to mid-bicellular pollen. Both promoter-barnase transgenes did not affect the sporophyte and were inherited through the female germline. These results show that both the NTM19 and Bp4 promoters are expressed only in the male germline, and that the NTM19 promoter is an excellent tool to direct high levels of transgene expression exclusively to the microspores. This may have important biotechnological applications.     

Catabolism of indole-3-acetic acid in chloroplast fractions from light-grown Pisum sativum L. seedlings

Abstract The catabolism of indole-3-acetic acid was investigated in chloroplast preparations and a crude enzyme fraction derived from chloroplasts of Pisum sativum seedlings. Data obtained with both systems indicate that indole-3-acetic acid undergoes decarboxylative oxidation in pea chloroplast preparations. An enhanced rate of decarboxylation of [1′-¹C]indole-3-acetic acid was obtained when chloroplast preparations were incubated in the light rather than in darkness. Results from control experiments discounted the possibility of this being due to light-induced breakdown of indole-3-acetic acid. High performance liquid chromatography analysis of [2′-¹⁴C]indole-3-acetic acid-fed incubates showed that indole-3-methanol was the major catabolite in both the chloroplast and the crude enzyme preparations. The identification of this reaction product was confirmed by gas chromatography-mass spectrometry when [²H₅]indole-3-methanol was detected in a purified extract derived from the incubation of an enzyme preparation with ³²H₅]indole-3-acetic acid.     

The effect of indole-3-acetylaspartic acid on adventitious root formation on bean cuttings

The influence of indole-3-acetylaspartic acid (IAAsp) on rooting of stem cuttings from bean plants (Phaseolus vulgaris L.) of different ages, cultivated at different temperatures (17°, 21° and 25°C) was studied and compared to that of indole-3-acetic acid (IAA). At a concentration of 10^–4 M, IAAsp only nonsignificantly stimulated adventitious root formation, approximately to the same level as IAA in all treatments. IAAsp at 5×10^–4 M further enhanced rooting, by up 200% of control values, with little influence of temperature conditions and stock plant age. This concentration of IAA usually stimulated rooting more than the conjugate. The largest differences between the effects of IAAsp and IAA occured at the highest cultivation temperature of 25°C where stock plant age also influenced the response. The number of roots produced in comparison with the control, was enhanced from 350% on cuttings from the youngest plants to more than 600% on cuttings from the oldest. In contrast to the conjugate, 5×10^–4 M IAA induced hypocotyl swelling and injury of the epidermis at the base of cuttings, in all treatments.