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     

Physiological and morphological characteristics during development of pedunculate oak (Quercus robur L.) zygotic embryos

The developmental stages of oak zygotic embryos (ZEs) are characterized here according to morphological and physiological features. Seeds were harvested from June to September in 1-week intervals. Excised embryos were classified into four stages of development by using growth parameters. For physiological characterization, endogenous levels of abscisic acid (ABA), indole-3-acetic acid (IAA), l-proline, starch content and water status were determined. The expression of the oak legumin storage protein gene was tested in immature cotyledonary ZEs before and after ABA treatment. The ABA levels of the embryos showed a significant peak during the intermediate stage of maturation (stage III) and then decreased again at the end of the late maturation phase (stage IV). Concomitant with ABA, the moisture content declined with the maximum embryo size. High IAA levels were found at the beginning of embryo enlargement as exponential growth occurred (stage II) but decreased during further development. Starch accumulated gradually in the course of maturation, whereas significant values were found in stage IV ZEs near shedding. Proline, on fresh weight basis, was high during stages I and II. Osmotic potential increased when, by rapid dry matter accumulation, stage II ZEs reached their maximum size during early intermediate development. Expression of precocious germination was higher on hormone-free medium, in particular, among stage II and stage III ZEs. Variations in phytohormone levels in combination with changes in tissue water status seem to be important factors for oak ZE development.     

Determination of unbound concentration of the novel anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid in human plasma by ultrafiltration followed by high-performance liquid chromatography with fluorimetric detection

The novel anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a highly protein bound drug with narrow therapeutic window. We report a simple HPLC method with fluorimetric detection for the determination of free DMXAA concentration in human plasma. Sample preparation involves the ultrafiltration of plasma by a Centrisart device for 30 min at 2000 g and extraction with acetonitrile: methanol mixture. The method was validated with respect to recovery, selectivity, linearity, precision, and accuracy. Calibration curves for DMXAA were constructed at the concentration range of 0.5–40 μM in blank plasma and phosphate buffer. The difference between the theoretical and calculated concentration and the relative standard deviation were less than 10% at all quality control (QC) concentrations. The HPLC method has been used for the analysis of preclinical studies.     

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.     

IAA, ABA, polyamines and free amino acids associated with zygotic embryo development of Ocotea catharinensis

The aim of this work was to study morphological and biochemical aspects during zygotic embryogenesis in O. catharinensis, by measuring changes in the endogenous concentrations of proteins, amino acids, polyamines (PAs), indole-3-acetic acid (IAA) and abscisic acid (ABA). Buffer-soluble and insoluble protein contents were determined by spectrometry, and amino acids, PAs, IAA and ABA concentrations were determined by high performance liquid chromatography. Total amino acid accumulation, predominantly asparagine, occurred when the embryo showed completely developed cotyledons, with posterior reduction in the mature embryo. This decrease in total amino acid concentration in the mature embryo may result from their use in storage␣as well as for LEA protein synthesis. Free putrescine (Put) concentration decreased, while free spermine (Spm) increased during embryo development. This suggest a role for Put in the initial phases of embryogenesis when high rates of cell division occur, while elevated concentration of Spm are essential from the middle to the end of embryo development, when growth is mainly due to cell elongation. An IAA peak in zygotic embryos occurred during initial development, suggesting a link between growth and cellular division as well as with the establishment of bilateral symmetry. ABA concentration declined during initial stages of development then increased at the mature embryo stage, suggesting a possible relationship with dormancy and recalcitrance characteristics. Our results show that changes in the phytohormones (IAA, ABA and PAs) concentrations in combination with amino acids are likely important factors determining the developmental stages of O.␣catharinensis zygotic embryos.     

High-throughput screening of potential inhibitors for the metabolism of the investigational anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid

By screening potential inhibitors of drug metabolism using the in vitro models, potential drug-drug interactions in vivo may be predicted with the use of appropriate pharmacokinetic principles. This study aimed to develop a rapid screening system using human liver microsomes to efficiently identify the potential inhibitors of DMXAA metabolism. Initial IC50 was estimated by using a two-point method, and then Ki values were determined if required and compared with those initial IC50 values. More than 100 compounds including known substrates and inhibitors of human uridine diphosphate glucuronosyltransferases (UGTs) and cytochrome P450 (CYP), anti-cancer drugs and xanthenone analogues were screened for their inhibitory effect on DMXAA glucuronidation and 6-methylhydroxylation in human liver microsomes. Both metabolites of DMXAA, DMXAA acyl glucuronide (DMXAA-G) and 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA), formed in human liver microsomes were quantitated by validated HPLC methods. The results indicated that there was a significant relationship (r2 = 0.966, P < 0.001) between the two-point IC50 values and the apparent Ki values for 20 compounds showing significant inhibitory effects on DMXAA metabolism, suggesting the usefulness of the two-point determination for the initial screening of compounds. This study has been completed using a strategy for rapid HPLC analysis and thus provided early access to detailed information for potential inhibitors of DMXAA metabolism and allows for further DMXAA-drug interaction studies.     

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.     

Hormonal changes in papaya seedlings subjected to progressive water stress and re-watering

Changes on abscisic acid (ABA), jasmonic acid (JA) and indole-3-acetic acid (IAA) levels were investigated in papaya seedlings (Carica papaya L.) cv. “Baixinho de Santa Amalia” under progressive water stress and subsequent rehydration. Also, the behaviour of leaf gas exchange and leaf growth was determined under stress condition. The results indicated that ABA and JA differ in their pattern of change under water stress. ABA continuously increased in leaves and roots during the whole period of stress whereas JA showed a sharp increase and a later decrease in both organs. Re-watering reduced rapidly (24 h) leaf and root ABA to control levels whereas the influence on JA levels could not be assessed. Drought and recovery did not alter IAA levels in leaf and root tissues of papaya seedlings. In addition, water stress reduced stomatal conductance, photosynthetic rate, transpiration rate, the percentage of attached leaves and leaf growth. Rehydration reverted in few days the effects of stress on leaf growth and gas exchange parameters. Overall, the data suggest that ABA could be involved in the induction of several progressive responses such as the induction of stomatal closure and leaf abscission to reduce papaya water loss. In addition, the pattern of accumulation of JA is compatible with a triggering signal upstream ABA. The unaltered levels of IAA could suggest a certain adaptive ability of papaya to maintain active physiological processes under progressive drought stress.     

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     

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.     

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.     

A high-throughput method for the quantitative analysis of indole-3-acetic acid and other auxins from plant tissue

To investigate novel pathways involved in auxin biosynthesis, transport, metabolism, and response, we have developed a high-throughput screen for indole-3-acetic acid (IAA) levels. Historically, the quantitative analysis of IAA has been a cumbersome and time-consuming process that does not lend itself to the screening of large numbers of samples. The method described here can be performed with or without an automated liquid handler and involves purification solely by solid-phase extraction in a 96-well format, allowing the analysis of up to 96 samples per day. In preparation for quantitative analysis by selected ion monitoring-gas chromatography-mass spectrometry, the carboxylic acid moiety of IAA is derivatized by methylation. The derivatization of the IAA described here was also done in a 96-well format in which up to 96 samples can be methylated at once, minimizing the handling of the toxic reagent, diazomethane. To this end, we have designed a custom diazomethane generator that can safely withstand high flow and accommodate larger volumes. The method for IAA analysis is robust and accurate over a range of plant tissue weights and can be used to screen for and quantify other indolic auxins and compounds including indole-3-butyric acid, 4-chloro-indole-3-acetic acid, and indole-3-propionic acid.     

丛枝菌根真菌对玉米和棉花内源激素的影响*

在温室盆栽条件下研究了丛枝菌根（Ａｒｂｕｓｃｕｌａｒ　ｍｙｃｏｒｒｈｉｚａｓ,ＡＭ）真菌：ＧｉｇａｓｐｏｒａｒｏｓｅａＮｉｃｏｌ．＆Ｓｃｈｅｎｃｋ、Ｇｌｏｍｕｓ　ｍｏｓｓｅａｅ（Ｎｉｃｏｌ．＆　Ｇｅｒｄ．）Ｇｅｒｄｅｍａｎｎ　＆Ｔｒａｐｐｅ和Ｇｌｏｍｕｓ　ｖｅｒｓｉｆｏｒｍｅ　（Ｋａｒｓｔｅｎ）Ｂｅｒｅｈ对玉米和棉花植株内源激素的影响。结果表明,ＡＭ真菌在正常供水和干旱条件下均能显著提高玉米和棉花植株叶片和根内玉米素、生长素和赤霉素的含量,并降低脱落酸的含量。在植物体内含磷量、生长量及其生长发育阶段等一致、仅存在接种与不接种唯一差异条件下,供试ＡＭ真菌同样能改变玉米和棉花植株内源激素的平衡状况。接种处理植株的脱落酸含量与气孔阻力呈正相关关系。表明玉米和棉花植株抗旱性和生长状况的改善与ＡＭ真菌改变内源激素的平衡状况有关。接种ＡＭ真菌的植株表现较强的抗旱性;其生长量也显著大于不接种的对照。ＧＩ．ｖｅｒｓｉｆｏｒｍｅ的效应最大。     

Hormones in zygotic and microspore embryos of Brassica napus

A number of studies have used microspore-derived embryos (MDEs) as amodel for examining a range of processes, including hormonal regulation ofembryo development. We examined the hormonal physiology of MDEs with theprimaryobjective of testing the validity of using the MDE system as a model forhormonally-regulated development in zygotic embryos, through late stages. To dothis we identified and quantified endogenous levels of abscisic acid (ABA),indole-3-acetic acid (IAA) and a number of gibberellins (GAs), includingGA₁₉, GA₂₀, GA₁ and GA₈ in bothMDEsand zygotic embryos. The presence of GA₁₉, together with itsC₁₉ metabolites indicates that the early-13 hydroxylation pathway isoperative in both embryo systems. Gibberellins A₄ and GA₉were also identified, thereby confirming the presence of the earlynon-hydroxylation pathway in B. napus MDEs and zygoticembryos. In general, the pattern of change of hormone (ABA, IAA, GA₁and GA₂₀) content per embryo through embryogenesis was similar forMDEs and zygotic embryos. Indole-3-acetic acid and GA₁ increased toamaximum at day 30 after culture (DAC) before decreasing. Abscisicacid levels increased to a maximum at day 35, and declined in zygoticembryos but not in MDEs. GA₂₀ increased to the final harvest atmaturity, or day 40. The absolute content (g/embryo) of each hormone, howeverwas appreciably lower (5- to 15- fold) in the MDEs. This was not the result ofdilution into surrounding medium for ABA or IAA; GA₁, however, didaccumulate in the medium. Although there were absolute quantitative differencesin the levels of IAA and ABA found in the two embryo systems, the similaritiesin the pattern of hormone changes suggests that the MDE system can serve as auseful model for examining the physiological roles of hormones duringembryogenesis.     

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.     

Changes in endogenous indole-3-acetic acid and some biochemical parameters during seed development in <Emphasis Type="Italic">Camellia sinensis</Emphasis> (L.) O. Kuntze

The role of endogenous indole-3-acetic acid (IAA), soluble proteins and RNA in the development of tea (Camellia sinensis (L). O. Kuntze) seeds was investigated in the present study. The state of continuum even at full maturity and lack of a clear end point to seed development as indicated by the persistence of appreciable contents of proteins at full maturity in all the seed parts further confirmed the ‘recalcitrant nature’ of the tea seeds. Unlike the orthodox seeds, the level of free IAA in tea embryos also remained high even at full maturity. The total RNA content remained high in the stages with high moisture content but declined with progressive decline in moisture content.     

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.