Rhizogenesis in Forsythia×intermedia and Syringa vulgaris; application of a simple internode experimental system

 Differences in rhizogenesis between easy-to-root Forsythia×intermedia Zab. cv. Lynwood and difficult-to-root Syringa vulgaris L. cv. Madame Lemoine were measured in an experimental system based upon internodal stem sections excised from axillary shoot cultures. Root induction in Syringa was distinctly polar, responding best to distal application of indole-3-butyric acid (IBA), whereas Forsythia was equally responsive to IBA applied at either end. Root initiation in Syringa declined rapidly from 73% to 32% over 48 h when the application of a 24-h pulse of IBA was delayed following excision of the internode; in Forsythia a smaller decline (93–70%) occurred over 144 h. Forsythia internodes were the more responsive, and developed roots after distal or proximal application of 3 μM IBA, whereas Syringa required the distal application of 30 μM IBA. Received: 22 March 2000 / Revision received: 5 June 2000 / Accepted: 5 June 2000     

Structure elucidation of acid reaction products of indole-3-carbinol: Detection in vivo and enzyme induction in vitro

The potency of indole-3-carbinol (I3C) to form condensation products under acidic aqueous conditions was studied. After identifying a known dimer, 3,3′-diindolylmethane (DIM), we elucidated the structures of two trimers also found in acid reaction mixtures: 5,6,11,12,17,18-hexahydrocyclonona[1,2-b:4,5-b′:7,8-b″]tri-indole (CTI), and 2,3-bis[3-indolylmethyl] indole (BII). The formation of these indole oligomers was shown to be pH dependent. The highest amounts of DIM and BII were formed in aqueous solutions having a pH value ranging from 4 to 5. No CTI could be detected at pH values above 4.5. In rats that received an oral dose of I3C we could detect DIM and BII in gastric contents, stomach tissue, small intestine and liver. No CTI could be detected in vivo after oral exposure to I3C. In in vitro experiments, using rat hepatocytes, the cytochrome P-450IA1 apoprotein level, 7-ethoxyresorufin O-deethylation activity (EROD) and DT-diaphorase activity (DTD) were markedly enhanced by DIM and CTI as well as BII.     

L- and D-tryptophan aminotransferases from maize coleoptiles

Two forms of L-tryptophan aminotransferases (L-TAT-1 and L-TAT-2) and one D-tryptophan aminotransferase (D-TAT) were separated from maize coleoptiles by using L- and D-tryptophan as amino group donors. The enzymes were partially purlfied by hydrophobic and gel filtration column chromatographies. L-TAT-1 and L-TAT-2 had similar properties, showing optimum pH at 8–9 and a high optimum temperature of 50–60 C for catalytic activity. As the amino group acceptor for these two enzymes, α-keto glutaric acid was more effective than pyruvic, oxaloacetic and glyoxylic acids. The molecular masses of L-TAT-1 and L-TAT-2 estimated by gel filtration were approximately 80 kDa and 45 kDa, respectively. D-TAT had an optimum pH similar to those of L-TATs, but the optimum temperature was conslderably lower (30 C). Pyruvic acid was an effective amino group acceptor for D-TAT, whereas oxaloacetic and α-keto glutaric acids were not. D-Cycloserine completely inhibited the activity. The molecular mass of D-TAT was approximately 55 kDa. These three TATs required pyridoxal-5-phosphate for their catalytic activities.     

Molecular cloning of the gene for indolepyruvate decarboxylase from Enterobacter cloacae

Summary Although indole-3-acetic acid (IAA) is a well-known plant hormone, the main IAA biosynthetic pathway from l-tryptophan (Trp) via indole-3-pyruvic acid (IPyA) has yet to be elucidated. Previous studies have suggested that IAA is produced by Enterobacter cloacae isolated from the rhizosphere of cucumbers and its biosynthetic pathway may possibly be the same as that in plants. To elucidate this pathway, the IAA biosynthetic gene was isolated from a genomic library of E. cloacae by assaying for the ability to convert Trp to IAA. DNA sequence analysis showed that this gene codes for only one enzyme and its predicted protein sequence has extensive homology with pyruvate decarboxylase in yeast and Zymomonas mobilis. Cell-free extracts prepared from Escherichia coli harboring this gene could convert IPyA to indole-3-acetaldehyde (IAAld). These results clearly show that this pathway is mediated only by indolepyruvate decarboxylase, which catalyzes the conversion of IPyA to IAAld.     

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.     

镉和增强紫外线-B辐射复合作用对大豆生长的影响

研究了Cd^2+和增强紫外线-B(UV-B)辐射以及二者复合胁迫(Cd+UV-B)对大豆生长、光合作用、抗氧化酶活性和吲哚乙酸(IAA)氧化酶活性的影响,结果表明,UV-B辐射对大豆生长较CA^2+有更明显的抑制作用,主要是降低了光合作用,生物量减小;抑制节间的分化和伸长,节间减少,株高降低。UV-B辐射对POD、SOD活性有显著诱导作用,而Cd^2+明显颉颃UV-B对POD活性的诱导并抑制IAA氧化酶活性．在复合作用下,植物体内IAA氧化酶和POD活性较UV-B单独作用下显著降低,这两种酶活性降低会引起植物体内IAA含量升高,同时光合作用略有升高,这是株高和生物量较UV-B作用下有所增加的重要原因,复合胁迫还增强了对根伸长生长的抑制作用,根长度较对照显著降低(P<0．05)。IAA氧化酶和POD活性变化以及光合强度变化与大豆株高和生物量变化密切相关,这也是复合胁迫影响大豆生长状况的重要因素。     

Auxin activity and molecular structure of 2-alkylindole-3-acetic acids

2-Methylindole-3-acetic acid (2-Me-IAA) is a known auxin, but its 2-ethyl homologue has been considered inactive. Here we show that the compound previously bioassayed as 2-ethylindole-3-acetic acid (2-Et-IAA) was, in fact, 3-(3-methylindol-2-yl)propionic acid. The proper 2-Et-IAA and its 2-(n-propyl) homologue (2-Pr-IAA) are prepared, unambiguously characterized, and their auxin activity is demonstrated in the Avena coleoptile-section straight-growth test. Their half-optimal concentrations are approximately the same as for 2-Me-IAA (2 × 10^–5mol L^–1), and hence about ten times larger than for unsubstituted indole-3-acetic acid (IAA) and its derivatives alkylated in positions 4, 5, 6 or 7. The optimal response elicited by 2-Et-IAA and 2-Pr-IAA is about half that observed for 2-Me-IAA. These characteristics place the three 2-alkyl-IAAs along the borderline between the classes of strong and weak auxins, thus corroborating the results of interaction similarity analysis, a mathematical approach based on the capability of auxin molecules to participate in non-bonding interactions with a generalized receptor protein. X-ray diffraction analysis shows no explicit structural features to be blamed for the decrease in auxin activity caused by attaching a 2-alkyl substituent to the IAA molecule; sterical interference of the 3-CH₂COOH group and the 2-alkyl moiety is barely recognizable in the crystalline state. Quantum-chemical calculations and molecular dynamics simulations suggest that 2-alkyl-IAAs, in the absence of crystal-packing restraints, prefer conformations with the CH₂-COOH bond tilted to the heterocyclic ring system. Substantially higher conformational energy (and hence lower abundance) is predicted for planar conformers which were previously shown to prevail for IAA and many of its derivatives substituted in the benzene moiety of the indole nucleus. This shift in the rotational preferences of the -CH₂COOH moiety may be one of the reasons for the reduced plant-growth promoting activity of 2-alkyl-IAAs.     

The influence of kinetin on the endogenous content of indoleacetic acid in swelling seeds of Phaseolus, Zea and Pinus and young plants of Phaseolus

Treatment of different plant materials, seeds of Phaseolus vulgaris, Zea mays and Pinus silvestris and young plants of Phaseolus, with kinetin increased the level of extractable IAA. For seeds this increase was most pronounced in bean seeds, which contained the lowest amount of endogenous IAA and cytokinins, and lower in maize seeds with high endogenous content of IAA and cytokinins. – For young bean plants the kinetin treatment significantly increased the extractable amounts of IAA from all parts of the plant, hypocotyls, cotyledons, epicotyls and primary leaves, when the cut plants were placed for 24 h in kinetin solution. For plants sprayed with kinetin solution only the primary leaves showed a significantly higher level of extractable IAA, which could be explained by the fact that the plants were growing very close together, so that the primary leaves received most of the kinetin during spraying.     

1-Naphtalene acetic acid induces indole-3-ylacetylglucose synthase in Zea mays seedling tissue

The enzyme indole-3-acetylglucose synthase (UDPG: indole-3-ylacetylglucosyl transferase) catalyzes the reaction: UDPG + IAA 1-O-IAGlc + UDP. The enzyme is abundantly present inimmature maize endosperm, but present in lesser amount in the endosperm ofgerminating kernels. Rabbit polyclonal antibodies, against purified IAGlcsynthase, easily visualize the presence of the enzyme protein in endosperm, butnot in vegetative tissue. However, after 4 to 8 h of incubation ofmesocotyl and coleoptile segments in 50 M 1-naphthalene aceticacid (NAA) solution, the IAGlc synthase protein is detectable by Western blotanalysis, and enzyme activity determined in whole tissue homogenate is alsoincreased. Induction of IAGlc synthase by NAA is inhibited by cycloheximide.     

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 kinetin on the level of an indoleacetylaspartic acid like substance in germinating seeds and young plants of Phaseolus vulgaris

Kinetin treatment increased the level of an extractable indoleacetyl conjugate, apparently indoleacetylaspartic acid (IAAsp) in germinating seeds and young plants of Phaseolus vulgaris L. cv. Alabaster. The level of this substance in extracts of treated seeds was always higher than that from water treated seeds irrespective of whether the seeds had been extracted 24, 48 or 72 h after the beginning of the treatment. In all parts of young bean plants treated with kinetin there was more of the substance than in the corresponding parts of water treated plants.     

菜豆茎韧皮部卸出的细胞途径以及激素的促进作用(英文)

通过缩小叶面积和去茎尖改变源库比率,以调节韧皮部卸出的途径,证明了韧皮部卸出的共质体与质外体途径的季节变化,和由对氯高汞苯磺酸所诱发的从质外体向共质体途径的转变,是与光合产物的输入有关。缩小叶面积而降低源库比率,能增加夏季生长植株茎韧皮部的质外体卸出,但对冬季生长植株无影响。去尖而增加源库比率,则促进共质体卸出。赤霉酸和激动素能促进共质体的横向转运,但对质外体转运无作用。当质外体为主要运输途径时,赤霉酸和激动素开启共质体途径。赤霉酸和激动素刺激光合产物,通过共质体从筛管一伴胞复合体向韧皮部薄壁纽胞输送,并可能在韧皮部薄壁细胞被动扩散到自由空间。由此可进一步说明蔗糖在激素处理部位自由空间的增加。     

The Determination of Phaseic Acid by Monoclonal Antibody-Based Enzyme immunoassay

A monoclonal antibody PA3-2-B3, IgG₁ (Λ) is described which specifically recognizes phaseic acid and shows very little cross-reactivity (0.14%) with abscisic acid or dihydrophaseic acid (0.88%). Based on this antibody, an enzyme immunoassay was developed which displays a linearity range from 15 pg to 3 ng of phaseic acid. Results obtained with this assay agree with those obtained by gas chromatography-electron capture detection. Using the novel enzyme immunoassay, as well as an established immunoassay for abscisic acid, levels of these two compounds in leaves of Phaseolus vulgaris were determined as a function of plant age, water stress, recovery from stress, and feeding of abscisic acid through the transpiration stream. The production of phaseic acid in a microsomal system from bean leaves was demonstrated. The results show a regulation of the plant's capacity to metabolize abscisic acid to phaseic acid as a function of water stress.     

Bacteria associated with orchid roots and microbial production of auxin

Associative bacteria of terrestrial (Paphiopedilum appletonianum) and epiphytic (Pholidota articulata) tropical orchids were investigated. Microbial community of epiphytic plant differed from that of the terrestrial one. Streptomyces, Bacillus, Pseudomonas, Burkholderia, Erwinia and Nocardia strains populated Paphiopedilum roots, whereas Pseudomonas, Flavobacterium, Stenotrophomonas, Pantoea, Chryseobacterium, Bacillus, Agrobacterium, Erwinia, Burkholderia and Paracoccus strains colonized Pholidota roots. Endophytic bacteria populations were represented with less diversity: Streptomyces, Bacillus, Erwinia and Pseudomonas genera were isolated from P. appletonianum, and Pseudomonas, Bacillus, and Flavobacterium genera were isolated from Ph. articulata. Microorganisms produced indole-3-acetic acid (IAA). Variations in its biosynthesis among the strains of the same genus were also observed. The highest auxin level was detected during the stationary growth phase. Biological activity of microbial IAA was proved by treatment of kidney bean cuttings with bacterial supernatants, revealing considerable stimulation of root formation and growth.     

Indole compounds released by the ectendomycorrhizal fungal strain MrgX isolated from a pine nursery

The production and metabolism of indole compounds in pure cultures of the ectendomycorrhizal strain MrgX, a common symbiont of Scots pine in forest nurseries, were investigated. Different indole compounds produced by this fungus were purified and identified by thin-layer chromatography, high-performance liquid chromatography and mass spectrometry. Indole-3-acetic acid (IAA) and indole-3-carboxylic acid were the most abundant. Although MrgX is able to synthesize IAA when cultivated on a medium without tryptophan, much higher IAA production was obtained when 1 mM tryptophan was added. Buffering of the medium at pH 5.8 was shown to be essential for IAA accumulation in the culture filtrate. In vitro IAA-synthesizing activity of the enzymes extracted from the mycelia of MrgX was also maximal when mycelia were grown in a buffered, tryptophan-supplemented medium. The hydrogen ion concentration strongly affected in vivo activity of IAA-synthesizing enzymes. This activity was rather weak at acid pH and was stimulated by increase in pH up to 8.5. These results and their possible significance for ectendo-mycorrhizal symbiosis are discussed with reference to the hormonal metabolism of ectomycorrhizal fungi and ectomycorrhizae.     

Growth and indole-3-acetic acid biosynthesis of Azospirillum brasilense Sp245 is environmentally controlled

Batch and fed batch cultures of Azospirillum brasilense Sp245 were conducted in a bioreactor. Growth response, IAA biosynthesis and the expression of the ipdC gene were monitored in relation to the environmental conditions (temperature, availability of a carbon source and aeration). A. brasilense can grow and produce IAA in batch cultures between 20 and 38 degrees C in a standard minimal medium (MMAB) containing 2.5 gl(-1)l-malate and 50 microgml(-1) tryptophan. IAA synthesis requires depletion of the carbon source from the growth medium in batch culture, causing growth arrest. No significant amount of IAA can be detected in a fed batch culture. Varying the concentration of tryptophan in batch experiments has an effect on both growth and IAA synthesis. Finally we confirmed that aerobic growth inhibits IAA synthesis. The obtained profile for IAA synthesis coincides with the expression of the indole-3-pyruvate decarboxylase gene (ipdC), encoding a key enzyme in the IAA biosynthesis of A. brasilense.     

Endogenous hormonal content and somatic embryogenic capacity of Corylus avellana L. cotyledons

Endogenous indole-3-acetic acid (IAA), abscisic acid (ABA) and cytokinins [zeatin (Z) zeatin riboside, dihydrozeatin, dihydrozeatin riboside, N⁶-isopentenyl adenine (iP) and N⁶-isopentenyladenine riboside] were evaluated in hazelnut (Corylus avellana L.) cotyledons of different developmental stage and genetic source for their somatic embryogenic capacity. There was an inverse correlation between the embryogenic potential of cotyledons and the degree of maturity of zygotic embryos, the first characteristic being associated with iP-type cytokinins and the second with Z-type cytokinins. Although the differences in total cytokinin, ABA and IAA contents between the cotyledons were small, the IAA/ABA and, mainly, the iP-type/Z-type cytokinin ratios were found to be two good indexes of the embryogenic competence of explants, suggesting that the endogenous hormonal balance is a very important factor defining the in vitro potential of hazelnut cotyledons. Received: 6 January 1997 / Revision received: 3 March 1997 / Accepted 1 April 1997     

Effects of auxin on wall polysaccharide composition and enzyme activity during extension-growth of Pellia (Bryophyta)

Auxin-induced changes in cell wall polysaccharide composition and enzyme activity of seta segments from the liverwort Pellia epiphylla (L.) Corda were studied using colorimetric, gas chromatographic, radioactive tracer, and viscometric techniques. Extension-growth of segments doubled in the presence of aqueous 10 μ M indole-3-acctic acid (IAA) ± 50 m M glucose. IAA-enhanced growth was accompanied by (1) enhanced synthesis of all wall polysaccharides but cellulose, (2) increase in the relative glucose content of neutral wall sugars, and (3) change in the activity of wall-bound glycosidase relative to controls, but no change in the activity of cellulase. Galactose and mannose (50 m M ) suppressed auxin enhancement of both growth and wall synthesis. These findings suggest that auxin-mediated extension-growth of Pellia setae is dependent upon the maintenance of non-cellulosic cell-wall synthesis.     

Metabolism of auxin in pine tissues: Indole-3-acetic acid conjugation

Incubation of sections of various tissues of Pinus pinea L. with a relatively low concentration (3.6 μM) of indole-3-acetic acid-2-¹⁴C (IAA) resulted in the formation of two major metabolites. The first, which has not been identified, seemed to be a polar acidic compound and the second was identified as indole-3-acetylaspartic acid (IAAsp). The polar acidic metabolite has been found to be the major metabolite in needles, shoot wood and roots, while IAAsp has been found to be the major metabolite in shoot bark. Increasing the concentration of IAA in the incubation medium resulted in an increase in the formation of a third metabolite which proved to be l-O-(indole-3-acetyl)-β-d -glucose (IAGlu) and a concomitant decrease in the amount of the polar acidic metabolite. This phenomenon was prominent particularly in needles. IAGlu was isolated from needles and IAAsp was isolated from shoot bark by means of polyvinylpolypyrrolidone column chromatography and preparative thin-layer chromatography. IAGlu was identified by comparison with authentic material by co-chromatography in three different solvent systems and by ¹H-nuclear magnetic resonance analysis. IAAsp was identified by comparison with authentic material by gas-liquid chromatography and ¹H-nuclear magnetic resonance analysis. Several aspects of formation, separation and isolation of IAA metabolites are discussed.     

Endogenous indoles and the biosynthesis and metabolism of indole-3-acetic acid in cultures of Rhizobium phaseoli

Gas chromatography-mass spectrometric analyses of purified extracts from cultures of Rhizobium phaseoli wild-type strain 8002, grown in a non-tryptophan-supplemented liquid medium, demonstrated the presence of indole-3-acetic acid (IAA), indole-3-ethanol (IEt), indole-3-aldehyde and indole-3-methanol (IM). In metabolism studies with ³H-, ¹⁴C- and ²H-labelled substrates the bacterium was shown to convert tryptophan to IEt, IAA and IM; IEt to IAA and IM; and IAA to IM. Indole-3-acetamide (IAAm) could not be detected as either an endogenous constituent or a metabolite of [³H]tryptophan nor did cultures convert [¹⁴C]IAAm to IAA. Biosynthesis of IAA in R. phaseoli, thus, involves a different pathway from that operating in Pseudomonas savastanio and Agrobacterium tumefaciens-induced crown-gall tumours.Abbreviations IAA indole-3-acetic acid - IAld indole-3-aldehyde - IAAm indole-3-acetamide - IEt indole-3-ethanol - IM indole-3-methanol - HPLC-RC high-performance liquid chromatography-radio counting - GC-MS gas chromatography-mass spectrometry