Differential expression and evolution of the Arabidopsis CYP86A subfamily

Some members of the Arabidopsis (Arabidopsis thaliana) CYP86A and CYP94B cytochrome P450 monooxygenase subfamilies, which share some sequence homology with the animal and fungal fatty acid hydroxylases, have been functionally defined as fatty acid omega-hydroxylases. With these activities, these and other fatty acid hydroxylases have potential roles in the synthesis of cutin, production of signaling molecules, and prevention of accumulation of toxic levels of free fatty acids. The constitutive and stress-inducible patterns of the five Arabidopsis CYP86A subfamily members have been defined in 7-d-old seedlings and 1-month-old plant tissues grown under normal conditions, and 7-d-old seedlings treated with different hormones (indole-3-acetic acid, abscisic acid, gibberellin, methyl jasmonic acid, brassinosteroid, salicylic acid), chemicals (clofibrate, 1-aminocyclopropane-1 carboxylic acid), or environmental stresses (cold, wounding, drought, mannitol, etiolation). Very distinct expression patterns exist for each of these fatty acid hydroxylases under normal growth conditions and in response to environmental and chemical stresses. Analysis of the promoter sequences for each of these genes with their expression patterns has highlighted a number of elements in current databases that potentially correlate with the responses of individual genes.     

The effect of selected plant hormones on in vitro proliferation of hyphae of Glomus fistulosum

Effects of N⁶-benzyladenine, kinetin, zeatin, N⁶-benzyladenosine, kinetin riboside, zeatin riboside, jasmonic acid, indole-3-acetic acid, indole-3-butyric acid, indole-3-propionic acid, abscisic acid and gibberellic acid on proliferation of hyphae of arbuscular mycorrhizal (AM) fungus Glomus fistulosum were studied under axenic conditions in vitro. The growth of intraradical hyphae of G. fistulosum was fully suppressed by 30 μM indole-3-acetic acid, but a perceptible decrease in the proliferation of the hyphae was observed already at 3 μM. Because such concentration is near the concentrations common in root tissues in vivo, the effect may be biologically significant. Similar effect was also observed for Glomus mosseae. Inhibitory effects of abscisic acid and cytokinins occurred only at very high, non-physiological concentrations. Ribosylated cytokinins showed stronger inhibition effects than their non-ribosylated counterparts. No stimulation of proliferation of hyphae by any plant hormone tested was observed. This revised version was published online in July 2006 with corrections to the Cover Date.     

A simple and efficient method for analysis of plant growth regulators: a new tool in the chest to combat recalcitrance in plant tissue culture

This report presents a simple, rapid and accessible validated method for quantification of eight major plant growth regulators (PGR): cytokinins (6-(γ,γ-dimethylallylamino)purine (2-iP), benzylaminopurine (BA) and zeatin), auxin (indole-3-acetic acid; IAA), jasmonic acid (JA), salicylic acid (SA), gibberellic acid (GA3) and abscisic acid (ABA) by liquid chromatography mass spectrometry. This method was tested in eight species including agricultural, ornamental and medicinal species: St. John’s wort, African violet, banana, American elm, tobacco, potato, sweet wormwood, and fennel. The method has good reproducibility and good sensitivity with %RSD (percent relative standard deviation) between 1 and 10% for all matrices and recovery values of 89 to 118% for all analytes. Method detection limits were 50.65 ng/g, 203.4 ng/g, 50.65, ng/g, 50.65 ng/g, 203.4 ng/g, 12.7 ng/g, 193 pg/g and 3.08 ng/g, for SA, IAA, zeatin, JA, GA3, ABA, 2-iP, and BA, respectively. Our results with a range of plant species show that this method represents a simple, low-cost method for analysis of PGRs, and may also serve as an useful starting point for the analysis of other related PGRs, as demonstrated by inclusion of the SA derivative, acetylsalicylic acid, and the JA derivatives: 12-oxo-phytodienoic acid and JA-isoleucine. The efficiency of this method will enable its incorporation into the plant tissue culture work flow and through characterization of endogenous PGR levels, will allow for improved method development for recalcitrant species facilitating fundamental and applied studies in plant morphogenesis, propagation and conservation.     

Simultaneous quantification of major phytohormones and related compounds in crude plant extracts by liquid chromatography-electrospray tandem mass spectrometry

A rapid and sensitive method was developed for simultaneous quantification of multiple classes of phytohormones and some related metabolites in crude plant extracts without purification or derivatization. High-performance liquid chromatography and electrospray ionization–tandem mass spectrometry with multiple reaction monitoring were used to quantify auxins, cytokinins, abscisic acid, gibberellins, jasmonates, salicylates, and a number of related metabolites in crude plant extracts. The technology was applied to analyze biotic and abiotic stress-induced changes of phytohormones in Arabidopsis tissues, starting with 50–100 mg fresh tissue. Biotic and/or abiotic stresses were shown to differentially affect levels of salicylic acid, jasmonic acid, indole-3-acetic acid, and benzoic acid, in comparison to their methyl esters. Compared with previous methods, sample preparation time and amount of sample required for analysis of phytohormones are reduced, and more classes of hormones are quantitatively profiled. Structurally diverse compounds from complicated biological matrices are determined with high selectivity and sensitivity.     

Increases in jasmonic acid caused by indole-3-acetic acid and auxin herbicides in cleavers (Galium aparine)

The effects of indole-3-acetic acid and auxin herbicides on endogenous jasmonic acid (JA) concentrations were studied in relation to changes in ethylene and abscisic acid (ABA) levels in cleavers (Galium aparine). When plants were root-treated with increasing concentrations of indole-3-acetic acid (IAA), ethylene biosynthesis was stimulated in response to the accumulation of endogenous IAA in the shoot tissue. Within 25h of treatment, stimulated ethylene formation was accompanied by increases in immunoreactive concentrations of JA and ABA, which reached maxima of 4.5-fold and 26-fold of the control, respectively, at 100 microM of applied IAA. Corresponding effects were obtained using synthetic auxins and when the ethylene-releasing compound ethephon was applied exogenously. This represents the first report, to our knowledge, of an auxin-mediated increase in JA levels. The increase in JA may be triggered by ethylene.     

Simultaneous quantification of phytohormones in fermentation extracts of Botryodiplodia theobromae by liquid chromatography–electrospray tandem mass spectrometry

Fermentation broth and biomass from three strains of Botryodiplodia theobromae were characterized by high performance liquid chromatography–electrospray tandem mass spectrometry (HPLC–ESI–MS/MS) method, in order to quantify different phytohormones and to identify amino acid conjugates of jasmonic acid (JA) present in fermentation broths. A liquid–liquid extraction with ethyl acetate was used as sample preparation. The separation was carried out on a C18 reversed-phase HPLC column followed by analysis via ESI–MS/MS. The multiple reaction monitoring mode was used for quantitative measurement. For the first time, indole-3-acetic acid, indole-3-propionic acid, indole-3-butyric acid and JA were identified and quantified in the ethyl acetate extracts from the biomass, after the separation of mycelium from supernatant. The fermentation broths showed significantly higher levels of JA in relation to the other phytohormones. This is the first report of the presence of gibberellic acid, abscisic acid, salicylic acid and the cytokinins zeatin, and zeatin riboside in fermentation broths of Botryodiplodia sp. The presence of JA-serine and JA-threonine conjugates in fermentation broth was confirmed using HPLC-ESI tandem mass spectrometry in negative ionization mode, while the occurrence of JA-glycine and JA-isoleucine conjugates was evidenced with the same technique but with positive ionization. The results demonstrated that the used HPLC–ESI–MS/MS method was effective for analysing phytohormones in fermentation samples.     

Biological activity and biosynthesis of pentacyclic oxylipins: The linoleic acid pathway

The relevance of the postulated pathway from linoleic acid to dihydrojasmonic acid is analysed. Pentacyclic oxylipins having pentenyl or pentyl side chains were tested for their secondary metabolite inducing activity in seven different plant cell culture species which all responded well to 12-oxo-phytodienoic acid and jasmonic acid. The response towards the dihydro-derivatives 15,16-dihydro-12-oxo-phytodienoic acid and 9,10-dihydrojasmonic acid ranged from strong activity in Eschscholzia californica to no activity in Lycopersicon esculentum. 15,16-Dihydro-12-oxo-phytodienoic acid can be formed from linoleic acid (18:2) by a linseed acetone powder enzyme preparation. Application experiments with linoleic (18:2) and linolenic acid (18:3) showed that the bottleneck of the 18:2 pathway is most likely the cyclization of the intermediate allene oxide when compared to the ease by which 15,16-dihydro-12-oxo-phytodienoic acid is converted to dihydrojasmonic acid in plant systems. The metabolism of potential precursors of jasmonic and dihydrojasmonic acid was extensively studied in various cell cultures.     

Biochemical characterization of enoyl reductase involved in Type II fatty acid synthesis in the intestinal coccidium Eimeria tenella (Phylum Apicomplexa)

Moniliophthora perniciosa is the causative agent of witches' broom disease in Theobroma cacao. Exogenously provided abscisic acid (ABA), indole-3-acetic acid (IAA), jasmonic acid (JA), and salicylic acid (SA) promoted mycelial growth, suggesting the ability of the pathogen to metabolize plant hormones. ABA, IAA, JA, and SA were found endogenously in the mycelium and in the fruiting body of the pathogen. The pathogen contained high amounts of SA in the mycelium (0.5+/-0.04 microg g(-1) DW) and IAA (2+/-0.6 microg g(-1) DW) in the basidiocarps. Growth of the mycelium in the presence of host leaves for 10 days did not affect ABA or JA content of the leaves but IAA and SA increased 2.5- and 11-fold, respectively. The amounts of IAA and SA in infected leaves increased beyond the levels of the uninfected leaves and suggest a synergistic response to host-pathogen interaction. The ability of M. perniciosa to produce and sustain growth in the presence of elevated endogenous IAA and SA levels during colonization indicates that these phytohormones contribute to its pathogenicity.     

Negative cross-talk between salicylate- and jasmonate-mediated pathways in the Wassilewskija ecotype of Arabidopsis thaliana

Plants often respond to attack by insect herbivores and necrotrophic pathogens with induction of jasmonate-dependent resistance traits, but respond to attack by biotrophic pathogens with induction of salicylate-dependent resistance traits. To assess the degree to which the jasmonate- and salicylate-dependent pathways interact, we compared pathogenesis-related protein activity and bacterial performance in four mutant Arabidopsis thaliana lines relative to their wild-type backgrounds. We found that two salicylate-dependent pathway mutants (cep1, nim1-1) exhibited strong effects on the growth of the generalist biotrophic pathogen, Pseudomonas syringae pv. tomato, whereas two jasmonate-dependent pathway mutants (fad3-2fad7-2fad8, jar1-1) did not. Leaf peroxidase and exochitinase activity were negatively correlated with bacterial growth, whereas leaf polyphenol oxidase activity and trypsin inhibitor concentration were not. Interestingly, leaf total glucosinolate concentration was positively correlated with bacterial growth. In the same experiment, we also found that application of jasmonic acid generally increased leaf peroxidase activity and trypsin inhibitor concentration in the mutant lines. However, the cep1 mutant, shown previously to overexpress salicylic acid, exhibited no detectable biological or chemical responses to jasmonic acid, suggesting that high levels of salicylic acid may have inhibited a plant response. In a second experiment, we compared the effect of jasmonic acid and/or salicylic acid on two ecotypes of A. thaliana. Application of salicylic acid to the Wassilewskija ecotype decreased bacterial growth. However, this effect was not observed when both salicylic acid and jasmonic acid were applied, suggesting that jasmonic acid negated the beneficial effect of salicylic acid. Collectively, our results confirm that the salicylate-dependent pathway is more important than the jasmonate-dependent pathway in determining growth of P. syringae pv. tomato in A. thaliana, and suggest important negative interactions between these two major defensive pathways in the Wassilewskija ecotype. In contrast, the Columbia ecotype exhibited little evidence of negative interactions between the two pathways, suggesting intraspecific variability in how these pathways interact in A. thaliana.     

外源茉莉酸甲酯诱导抗性与敏感稗草对二氯喹啉酸抗性的差异及机理

以遗传背景一致的抗二氯喹啉酸型和敏感型稗草为供试材料,分析了经外源茉莉酸甲酯(MeJA)预处理后,稗草种群间应对二氯喹啉酸胁迫的抗性水平及4种内源激素含量的变化。结果表明: 外源MeJA处理可显著提高抗性稗草对二氯喹啉酸的抗性,而对敏感稗草的抗性影响不显著。供试稗草间植株体内生长素(IAA)、脱落酸(ABA)、水杨酸(SA)和茉莉酸(JA)的含量及其变化幅度存在显著差异。二氯喹啉酸处理显著增加了稗草ABA、SA和JA的含量,且敏感稗草ABA和JA含量升高幅度明显高于抗性稗草;外源MeJA预处理能显著增强二氯喹啉酸对稗草ABA和JA含量的诱导,且敏感稗草变化幅度较大。表明敏感稗草较快的激素变化不利于其应对除草剂胁迫,抗性稗草相对平缓的激素变化赋予了其对除草剂的适应性,且MeJA处理增强了其对二氯喹啉酸的抗性。稗草植株体内ABA和JA激素信号在其抵抗二氯喹啉酸胁迫过程中可能发挥重要的生物学功能。     

Visualization of dynamics of plant-pathogen interaction by novel combination of chlorophyll fluorescence imaging and statistical analysis: differential effects of virulent and avirulent strains of P. syringae and of oxylipins on A. thaliana

Pathogen infection leads to defence induction as well as to changes in carbohydrate metabolism of plants. Salicylic acid and oxylipins are involved in the induction of defence, but it is not known if these signalling molecules also mediate changes in carbohydrate metabolism. In this study, the effect of application of salicylic acid and the oxylipins 12-oxo-phytodienoic acid (OPDA) and jasmonic acid on photosynthesis was investigated by kinetic chlorophyll fluorescence imaging and compared with the effects of infection by virulent and avirulent strains of Pseudomonas syringae. Both pathogen strains and OPDA caused a similar change in fluorescence parameters of leaves of Arabidopsis thaliana. The response to OPDA appeared faster compared with that to the pathogens and persisted only for a short time. Infiltration with jasmonic acid or salicylic acid did not lead to a localized and distinct fluorescence response of the plant. To capture the faint early symptoms of the plant response, a novel algorithm was applied identifying the unique fluorescence signature-the set of images that, when combined, yield the highest contrast between control and infected leaf segments. Unlike conventional fluorescence parameters, this non-biased approach indeed detected the infection as early as 6 h after inoculation with bacteria. It was posssible to identify distinct fluorescence signatures characterizing the early and late phases of the infection. Fluorescence signatures of both infection phases were found in leaves infiltrated with OPDA.     

Activation of the indole-3-acetic acid-amido synthetase GH3-8 suppresses expansin expression and promotes salicylate- and jasmonate-independent basal immunity in rice

New evidence suggests a role for the plant growth hormone auxin in pathogenesis and disease resistance. Bacterial infection induces the accumulation of indole-3-acetic acid (IAA), the major type of auxin, in rice (Oryza sativa). IAA induces the expression of expansins, proteins that loosen the cell wall. Loosening the cell wall is key for plant growth but may also make the plant vulnerable to biotic intruders. Here, we report that rice GH3-8, an auxin-responsive gene functioning in auxin-dependent development, activates disease resistance in a salicylic acid signaling- and jasmonic acid signaling-independent pathway. GH3-8 encodes an IAA-amino synthetase that prevents free IAA accumulation. Overexpression of GH3-8 results in enhanced disease resistance to the rice pathogen Xanthomonas oryzae pv oryzae. This resistance is independent of jasmonic acid and salicylic acid signaling. Overexpression of GH3-8 also causes abnormal plant morphology and retarded growth and development. Both enhanced resistance and abnormal development may be caused by inhibition of the expression of expansins via suppressed auxin signaling.     

Partial isotope fractionation during high-performance liquid chromatography of deuterium-labelled internal standards in plant hormone analysis: A cautionary note

Deuterium-labelled indole-3-acetic acid, abscisic acid and phthalimido-1-aminocyclopropane-1-carboxylic acid were found to separate from the unlabelled compounds on reverse-phase high-performance liquid chromatography (HPLC). A similar separation was found for the methyl esters of these compounds on normal-phase HPLC. Such separations may lead to substantial errors when these compounds are used as internal standards for quantitation by gas chromatography-mass spectrometry/selective ion detection, unless the complete chromatographic peaks are collected.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - IAA indole-3-acetic acid - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - Pht-ACC phthalimido-ACC - SIM selected ion monitoring     

An advanced method for the determination of carboxyl methyl esterase activity using gas chromatography-chemical ionization-mass spectrometry

We developed a quantitative method for the determination of methyl esterase activity, analyzing substrate specificity against three major signal molecules, jasmonic acid methyl ester (MeJA), salicylic acid methyl ester (MeSA), and indole-3-acetic acid methyl ester (MeIAA). We used a silylation reagent for chemical derivatization and used gas chromatography (GC)-mass spectroscopy in analyses, for high precision. To test this method, an Arabidopsis esterase gene, AtME8, was expressed in Escherichia coli, and then the kinetic parameters of the recombinant enzyme were determined for three substrates. Finally, this method was also applied to the direct quantification of phytohormones in petals from lilies and roses.     

Hormone homeostasis and induction of the small-fruit phenotype in ‘Hass’ avocado

Plant hormones play an important regulatory role in growth and development of fleshy fruits and often these are synthesized in a sequence that is tissue- and/or fruit-specific. Previous studies using Hass avocado and its small-fruit phenotype as a model demonstrated a relationship between the cytokinin/abscisic acid ration and final fruit size. More detailed studies revealed that differential activity of molybdenum-cofactor-containing enzymes was in part responsible for the observed increase in abscisic acid in small Hass fruit and that the same suite of enzymes likely impacted levels of indole-3-acetic acid. The present study confirms that the inability of the seed to produce/accumulate indole-3-acetic acid is a major contributing factor and characteristic of fruit that display a small-fruit phenotype, that reduced indole-3-acetic acid levels occur concomitant with increased abscisic acid, and that cytokinin metabolism appears unchanged irrespective of fruit size. Results are discussed in relation to the role of molybdenum-cofactor-requiring enzymes in indole-3-acetic acid and abscisic acid metabolism.     

Determination of phytohormones in phloem exudate from tree species by radioimmunoassay

A sensitive and specific radioimmunoassay was used to determine quantitatively four of the most important phytohormones in the phloem exudate from 14 different tree species of 8 genera. For cytokinins and indole-3-acetic acid (IAA) we found higher concentrations than those reported previously for other species. The gibberellin values were of the same order of magnitude as in earlier analyses (with different methods) of tree phloem exudates, but lower than the ones reported for Ricinus. Free abscisic acid (ABA) was found in tree phloem exudates in similar concentrations as before in Yucca or palm phloem exudate, but at considerably lower ones than reported for Ricinus and in Lupinus phloem exudate.Abbreviations IAA indole-3-acetic acid - ABA abscisic acid - GA gibberellic acid     

Genome‐wide identification,expression analysis of auxin‐responsive GH3 family genes in maize (Zea mays L.) under abiotic stresses

Auxin is involved in different aspects of plant growth and development by regulating the expression of auxinresponsive family genes. As one of the three major auxinresponsive families, GH3(Gretchen Hagen3) genes participate in auxin homeostasis by catalyzing auxin conjugation and bounding free indole-3-acetic acid(IAA) to amino acids.However, how GH3 genes function in responses to abiotic stresses and various hormones in maize is largely unknown.Here, the latest updated maize(Zea mays L.) reference genome sequence was used to characterize and analyze the Zm GH3 family genes from maize. The results showed that 13 Zm GH3 genes were mapped on fi ve maize chromosomes(total10 chromosomes). Highly diversi fi ed gene structures and tissue-speci fi c expression patterns suggested the possibility of function diversi fi cation for these genes in response to environmental stresses and hormone stimuli. The expression patterns of Zm GH3 genes are responsive to several abiotic stresses(salt, drought and cadmium) and major stress-relatedhormones(abscisic acid, salicylic acid and jasmonic acid)Various environmental factors suppress auxin free IAA contents in maize roots suggesting that these abiotic stresses and hormones might alter GH3-mediated auxin levels. The respon siveness of Zm GH3 genes to a wide range of abiotic stresses and stress-related hormones suggested that Zm GH3 s are involved in maize tolerance to environmental stresses.     

Carriers for abscisic acid and indole-3-acetic acid in primary roots: their regional localisation and thermodynamic driving forces

A carrier for the uptake of abscisic acid (ABA) is present in the tips and elongating zones of primary roots of both leguminous (runner bean, French bean, pea) and non-leguminous (sunflower, maize) seedlings. No ABA carrier was present in more mature root regions. For indole-3-acetic acid both carrier-mediated uptake and a 2,3,5-triiodobenzoate-sensitive efflux component are present in growing and in non-elongating runner-bean root tissues. Both ABA and indole-3-acetic acid carriers were inactivated by protein-modifying reagents. The driving forces for the carrier systems were studied using reagents, (KCl, fusicoccin, vanadate, dicyclohexylcarbodiimide, proton ionophores and azide) known to modify transmembrane pH (ΔpH) and electricla gradients (ΔE) and whose effects were independently monitored using radiolabelled, lipophilic, weak acids as probes. For abscisic acid the carrier-mediated uptake depend on ΔpH and the nonsaturable component of uptake, due to diffusion of undissociated ABA. The maximum velocity of the carrier is greater at pH 4 than at pH 5, although the Michaelis constants are similar. Modification of ΔE did not alter ABA net uptake but effects on the indole-3-acetic acid system consistent with perturbation of an electrogenic 2,3,5-triiodobenzoate-sensitive component were observed. It is suggested that the ABA carrier is an ABA anion/hydrogen ion symport or, less likely, represents facilitated diffusion of undissociated ABA.