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.     

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.     

Effect of exogenous arginine on sugarcane ( Saccharum sp.) somatic embryogenesis, free polyamines and the contents of the soluble proteins and proline

The aim of this study was to evaluate the effect of arginine on sugarcane (Saccharum sp.) somatic embryogenesis, free polyamines and other nitrogenous compounds contents. Segments of leaves were used as explants to establish embryogenic cultures on media with 0 and 50.0 mg l⁻¹ arginine. Somatic embryos formation and free polyamines, free proline and total soluble proteins contents were compared. Arginine significantly induced sugarcane somatic embryogenesis. Free proline and protein levels determined in embryogenic cell masses during embryo differentiation-maturation, showed an arginine-induced promotion associated to the enhancement of the embryogenic process. In addition, free putrescine and, in a minor extent, spermidine and spermine contents were enhanced by arginine.     

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     

Immunomodulation of ABA function affects early events in somatic embryo development

 Immunomodulation of abscisic acid (ABA) function during somatic embryogenesis of Nicotiana plumbaginifolia has been used to demonstrate for the first time the effect of this phytohormone on early embryonic events. A homozygous transgenic line constitutively expressing an anti-abscisic acid (ABA) single chain fragment variable antibody in the endoplasmic reticulum was established. Development of somatic embryos from the transgenic line and the wild type was compared. The ABA biosynthesis mutants aba1 and aba2 and wild type cultures treated with the ABA biosynthesis inhibitor fluridone were also used for the comparative investigations. The development of embryonic structures was disturbed in the early stages of all cultures in which ABA function was blocked or which were ABA-deficient. After ABA complementation of the in vitro cell cultures normal somatic embryo development was restored. Received: 23 May 2000 / Revision received: 1 September 2000 / Accepted: 4 September 2000     

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.     

Protein and free amino acids in field-grown cowpea seeds as affected by water stress at various growth stages

Summary This study was undertaken to evaluate water stress effects during vegetative, flowering, and podfilling stages of cowpea plants (Vigna unguiculata L.) grown under natural field conditions in southern California on seed yield and protein and free amino acid content of the cowpea seeds. The lowest concentration of N was found in the seeds of the control treatment plants while the seed yield from these treatments was the highest as compared with the N concentration and yield of seeds from plants subjected to water stress during flowering and podfilling stages. The concentration of N in the seeds was inversely related to the seed dry weight yield. Protein arginine,-threonine,-serine,-cystine,-valine,-methionine, and-isoleucine were significantly affected by water stress at the three growth stages. There was no consistent pattern in the effect of water stress on the individual amino acids. The sum of protein amino acids in the cowpea seeds was not significantly influenced by the various treatments since some of the protein amino acids increased and others decreased producing an averaging effect on the figures comprising the sums of the amino acids. Water stress during the flowering and pod-filling stages increased the free amino acid pool, and at the same time, inhibited incorporation of the amino acids into the protein chain-thus lowering the protein amino acid fraction simultaneously. With the exception of methionine plus cystine, the essential amino acids in the seeds were present at concentrations equal to or greater than recommended by the World Health Organization and FAO. It is of particular importance to note that the concentration of lysine in the cowpeas was substantially higher than that found in wheat grain. It is also important to note that the amount of essential amino acids per gram of protein was not measurably affected by the water stress treatments during any of the growth stages.     

Distribution and transport of IAA in tomato plants

We determined as to indole-3-acetic acid (IAA), endogenous levels byliquid chromatography-mass spectrometry using ¹³C₆-IAA, diffusible levels byfluorometric detection using indole-propionic acid, and polar transportactivityby radioactive IAA in 3-month-old tomato plants (stems, leaves or roots). TheIAA concentration in the apoplast (AP) solution was higher than those in thesymplast (SP) solution in both the upper and lower parts of stems, showing thatIAA analysis of AP solution is important. Younger leaves exported much morediffusible IAA than older leaves. The IAA concentration in the main roots wasalmost at the same level as in the stems. The results suggested that thetransport capacity of IAA is probably the limiting factor for the amount of IAAtransported in stems and the amount of polar IAA transport might be only 19% ofthe endogenous IAA amount in stems.     

Protein and free amino acids in wheat grain as affected by soil types and salinity levels in irrigation water

Summary The effects of four lysimeter soil series under three salinity levels were evaluated for grain yield, wt/1000 seeds, protein, and amino acids in Mexican dwarf wheat (Triticum aestivum L. var. Cajeme 71). The soil series consisted of: Holtville clay loam, Greenfield sandy loam, San Emigdio sandy loam, and Altamont clay loam. The irrigation water salinity levels were designated: low –2.2 mmho, medium –4.2 mmho, and high –7.1 mmho.No significant differences were found in the amount of grain harvested or wt/1000 seeds in the 1976 crop produced on the differential soil series. The yield of the 1977 crop was significantly affected by the soil types.Effects of soil type on the protein amino acids in the grain in both years were similar. Significantly higher protein amino acid levels of histidine, arginine, aspartic acid, threonine, serine, glutamic acid, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine were found in the grain grown on Altamont clay loam soil than the other types.The free amino acids in grain from the 1976 and 1977 crops were similarly affected by the soil types, except that the quantitative values of the free amino acids were substantially lower in 1977 than in 1976. The free amino acids significantly influenced by soil types were tryptophane, lysine, arginine, aspartic acid, threonine, serine, glycine, alanine, valine, isoleucine, tyrosine, and phenylalanine. In both years' crops, the sum of the free amino acid fractions was significantly higher in the grain produced on the Altamont soil than on the other soils.Salinity level in the irrigation water did not affect the 1976 crop yield or wt/1000 seeds. Although yields of the 1977 crop were significantly reduced by salinity, the wt/1000 seeds was not. The sum of protein amino acids was significantly higher in the 1976 and 1977 grain crops irrigated with high salinity water than in low salinity irrigated crops.An increased salinity irrigation water significantly reduced the sum of free amino acid fractions in the 1976 grain crop. Since some of the free amino acids in the 1977 grain crop increased while the others decreased due to the salinity level in the irrigation water, the sum of the free amino acid fractions was not significantly influenced.Significant interactions were found between soil types and salinity levels on free arginine, threonine, serine, glutamic acid, and alanine, and also on the sum of the free amino acids in the 1976 wheat grain. In the 1977 wheat grain, there were significant interactions between soil types and salinity levels on the free glutamic acid, valine, leucine, tyrosine, and phenylalanine, and on protein serine, glutamic acid, glycine, alanine, and the sum of the protein amino acids.The amounts of essential amino acids expressed as mg of amino acid/g of protein were not affected by the soil types or salinity levels. With the exception of lysine, and possibly threonine and methionine plus cystine, the essential amino acids were present in the grain at concentrations equal to or greater than recommended by WHO and FAO.     

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.     

Synthesis of symmetric and asymmetric diamides of citric acid and amino acids

Summary A convenient method for the synthesis of symmetric and asymmetric diamides of amino acids including DOPA and citric acid from 2-tert-butyl-1,3-di(N-hydroxysuccinimidyl)citrate and 1-tert-butyl-2,3-di(N-hydroxysuccinimidyl)citrate is described.Abbreviations AcOtBu tert-butyl acetate - i-Bu iso-butyl - tBu tert-butyl - Bzl benzyl - p-OH-Bzl p-hydroxybenzyl - m,p-(OH)₂-Bzl m,p-dihydroxybenzyl - DCCI dicyclohexylcarbodiimide - Et ethyl - Me methyl - Su succinimidyl - SuOH N-hydroxysuccinimide - Ph phenyl     

Biosynthesis, oxidation and conjugation of aliphatic polyamines in higher plants

Summary. This chapter will focus on polyamine biosynthesis, oxidation, conjugation processes, mainly to hydroxycinnamic acids, and compartmentation of enzymes, substrates and products, giving an overview about recent results especially in higher plants. New research advances regarding the cloning of the main cDNA encoding for polyamine biosynthetic and oxidative enzymes, will be taken into consideration. Received January 4, 2000 Accepted March 7, 2000     

Optimization of a free separation of 30 free amino acids and peptides by capillary zone electrophoresis with indirect absorbance detection: a potential for quantification in physiological fluids

This report describes a rapid, single-run procedure, based on the optimization of capillary electrophoresis (CE) and indirect absorbance detection capabilities, which was developed for the separation and quantification of 30 underivatized physiological amino acids and peptides, usually present in biological fluids. p-Aminosalicylic acid buffered with sodium carbonate at pH 10.2+/-0.1 was used as the running electrolyte. Electrophoresis, carried out in a capillary (87 cm x 75 microm) at 15 kV potential (normal polarity), separated the examined compounds within 30 min. Limits of detection ranged from 1.93 to 20.08 micromol/l (median 6.71 micromol/l). The method was linear within the 50-200 micromol/l concentration range (r ranged from 0.684 to 0.989, median r=0.934). Within run migration times precision was good (median C.V.=0.7%). Less favorable within run peak area precision (median C.V.=6.6%) was obtained. The analytical procedure presented was successfully tested for separation and quantification of amino acids in physiological fluids, such as plasma or supernatant of macrophage cultures. Sample preparations require only a protein precipitation and dilution step.     

Phytomonas: transport of amino acids, hexoses and polyamines

Phytomonas cells (Phytomonas Jma) isolated from the latex of Jatropha macrantha were assayed for amino acid, hexose and polyamine transport. Results showed high transport rates for glucose and fructose (193 and 128 pmol min(-1) 10(-7) cells, respectively) and lower, but significant rates, for proline, arginine, cysteine and glutamate (between 1.7 and 5.8 pmol min(-1) 10(-7) cells). Minor transport activities were observed for serine, glycine and aspartate (<1 pmol min(-1) 10(-7) cells). Amino acid transport processes do not seem to be regulated by starvation or during the growth phases. Polyamine transport was also evaluated showing a clear preference for spermidine over putrescine (3.4 and 0.4 pmol min(-1) 10(-7) cells, respectively). This work represents the first report on metabolite transport in phytomonads.     

Dynamics of the concentration of IAA and some of its conjugates during the induction of somatic embryogenesis in Coffea canephora

Most of the somatic embryogenesis (SE) process requires the presence, either before or during the embryogenic process, of at least one exogenous auxin. This exogenous auxin induces the presence of endogenous auxins, which appears to be essential for SE induction. We found that during the preincubation period of SE in Coffea canephora, there is an important increase in both free and conjugated indole-3-acetic acid (IAA), as well as indole-3-butyric acid. This increase is accompanied by an increase in the expression of YUCCA (CcYUC), TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (CcTAA1), and GRETCHEN HAGEN 3 (GH3) genes. On the other hand, most of the IAA compounds decreased during the induction of SE. The results presented in this research suggest that a balance between free IAA and its amide conjugates is necessary to allow the expression of SE-related genes.     

Simultaneous Determination of Indole-3-Acetic Acid and Abscisic Acid as the Pentafluorobenzyl Derivative with Electron-Capture Gas Chromatography

Indole-3-acetic acid (IAA) and abscisic acid (ABA) were converted in to pentafluorobenzyl esters by α-bromo-2, 3, 4, 5, 6-pentafluorotoluene at 55℃. The derivatization took about 90 minutes. The two esters generated were able to be simultaneously determined with electron-capture gas chromatography. The method is simple and sensitive. The minimal test does: 10-14g for IAA and 10-13g for ABA.     

Conformational fluctuations versus constraints in amino acid side chains: the evolution of information content from free amino acids to proteins

Like all other complex biological systems, proteins exhibit properties not found in free amino acids (i.e., emergent properties). Here, we explore top-down constraints experienced by the residue side chains in proteins compared to amino acids in increasingly complex molecular environments: free amino acids, end-capped amino acids, and the central residue in an alpha-helical nonapeptide. The crystalline structure of the contractile protein profilin Ib and the enzyme trypsin were chosen as objects of study, and submitted to 10 ns molecular dynamics (MD) simulations. The results revealed increased conformational constraints on the side chains when going from the simpler to the more complex compounds. A Shannon entropy (SE) analysis of the conformational behavior of the side chains showed in most cases a progressive and marked decrease in the SE of the chi1 and chi2 dihedral angles. This is equivalent to stating that conformational constraints on the side chain of residues increase their information content and, hence, recognition specificity compared to free amino acids. In other words, the vastly increased information content of a protein relative to its free monomers is embedded not only in the tertiary structure of the backbone, but also in the conformational behavior of the side chains. The postulated implication is that both backbone and side chains, by virtue of being conformationally constrained, contribute to the protein's recognition specificity toward other macromolecules and ligands.