Biosynthesis of indole-3-acetic acid in Azospirillum brasilense. Insights from quantum chemistry.
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Quantum chemical methods AM1 and PM3 and chromatographic methods were used to qualitatively characterize pathways of bacterial production of indole-3-acetic acid (IAA). The standard free energy changes (delta G(o)'sum) for the synthesis of tryptophan (Trp) from chorismic acid via anthranilic acid and indole were calculated, as were those for several possible pathways for the synthesis of IAA from Trp, namely via indole-3-acetamide (IAM), indole-3-pyruvic acid (IPyA), and indole-3-acetonitrile (IAN). The delta G(o)'sum for Trp synthesis from chorismic acid was -402 (-434) kJ.mol-1 (values in parentheses were calculated by PM3). The delta G(o)'sum for IAA synthesis from Trp were -565 (-548) kJ.mol-1 for the IAN pathway, -481 (-506) kJ.mol-1 for the IAM pathway, and -289 (-306) kJ.mol-1 for the IPyA pathway. By HPLC analysis, the possibility was assessed that indole, anthranilic acid, and Trp might be utilized as precursors for IAA synthesis by Azospirillum brasilense strain Sp 245. The results indicate that there is a high motive force for Trp synthesis from chorismic acid and for IAA synthesis from Trp, and make it unlikely that anthranilic acid and indole act as the precursors to IAA in a Trp-independent pathway. (+info)
Formation of lipoxygenase-pathway-derived aldehydes in barley leaves upon methyl jasmonate treatment.
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In barley leaves, the application of jasmonates leads to dramatic alterations of gene expression. Among the up-regulated gene products lipoxygenases occur abundantly. Here, at least four of them were identified as 13-lipoxygenases exhibiting acidic pH optima between pH 5.0 and 6.5. (13S,9Z,11E,15Z)-13-hydroxy-9,11,15-octadecatrienoic acid was found to be the main endogenous lipoxygenase-derived polyenoic fatty acid derivative indicating 13-lipoxygenase activity in vivo. Moreover, upon methyl jasmonate treatment > 78% of the fatty acid hydroperoxides are metabolized by hydroperoxide lyase activity resulting in the endogenous occurrence of volatile aldehydes. (2E)-4-Hydroxy-2-hexenal, hexanal and (3Z)- plus (2E)-hexenal were identified as 2,4-dinitro-phenylhydrazones using HPLC and identification was confirmed by GC/MS analysis. This is the first proof that (2E)-4-hydroxy-2-hexenal is formed in plants under physiological conditions. Quantification of (2E)-4-hydroxy-2-hexenal, hexanal and hexenals upon methyl jasmonate treatment of barley leaf segments revealed that hexenals were the major aldehydes peaking at 24 h after methyl jasmonate treatment. Their endogenous content increased from 1.6 nmol.g-1 fresh weight to 45 nmol.g-1 fresh weight in methyl-jasmonate-treated leaf segments, whereas (2E)-4-hydroxy-2-hexenal, peaking at 48 h of methyl jasmonate treatment increased from 9 to 15 nmol.g-1 fresh weight. Similar to the hexenals, hexanal reached its maximal amount 24 h after methyl jasmonate treatment, but increased from 0.6 to 3.0 nmol.g-1 fresh weight. In addition to the classical leaf aldehydes, (2E)-4-hydroxy-2-hexenal was detected, thereby raising the question of whether it functions in the degradation of chloroplast membrane constituents, which takes place after methyl jasmonate treatment. (+info)
Molecular cloning and ethylene-inducible expression of Chib1 chitinase from soybean (Glycine max (L.) Merr.).
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A soybean seed-specific PR-8 chitinase, named Chib2, has a markedly extended C-terminal segment compared to other plant Chib1 homologues of the PR-8 chitinase family known to date. To further characterize the molecular structure and the expression pattern of this chitinase family, we cloned two typical Chib1-similar cDNAs (Chib1-1 and Chib1-2) from soybeans by PCR-cloning techniques. The deduced primary sequence of Chib1-1 chitinase is composed of a signal peptide segment (26 amino acid residues) and a mature 273 amino acid sequence (calculated molecular mass 28,794, calculated pI 3.7). This Chib1-1 enzyme is more than 90% identical to Chib1-2 chitinase but is below 50% identical to Chib2 enzyme. Thus, we confirmed the occurrence of two distinct classes, Chib1 and Chib2 in the plant PR-8 chitinase family. The Chib1 genes, interrupted by one intron, were found to be up-regulated in response to ethylene in stems and leaves, but scarcely expressed in developing soybean seeds. Chib1 chitinases may be responsible for protecting the plant body from various pathogenic attacks. (+info)
An allele of the ripening-specific 1-aminocyclopropane-1-carboxylic acid synthase gene (ACS1) in apple fruit with a long storage life.
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An allele of the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene (Md-ACS1), the transcript and translated product of which have been identified in ripening apples (Malus domestica), was isolated from a genomic library of the apple cultivar, Golden Delicious. The predicted coding region of this allele (ACS1-2) showed that seven nucleotide substitutions in the corresponding region of ACS1-1 resulted in just one amino acid transition. A 162-bp sequence characterized as a short interspersed repetitive element retrotransposon was inserted in the 5'-flanking region of ACS1-2 corresponding to position -781 in ACS1-1. The XhoI site located near the 3' end of the predicted coding region of ACS1-2 was absent from the reverse transcriptase-polymerase chain reaction product, revealing that exclusive transcription from ACS1-1 occurs during ripening of cv Golden Delicious fruit. DNA gel-blot and polymerase chain reaction analyses of genomic DNAs showed clearly that apple cultivars were either heterozygous for ACS1-1 and ACS1-2 or homozygous for each type. RNA gel-blot analysis of the ACS1-2 homozygous Fuji apple, which produces little ethylene and has a long storage life, demonstrated that the level of transcription from ACS1-2 during the ripening stage was very low. (+info)
The auxin-binding protein Nt-ERabp1 alone activates an auxin-like transduction pathway.
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Hyperpolarization of tobacco protoplasts is amongst the earliest auxin responses described. It has been proposed that the auxin-binding protein, ABP1, or a related protein could be involved in the first step of auxin perception at the plasma membrane. Using for the first time homologous conditions for interaction between the protein Nt-ERabp1 or a synthetic peptide corresponding to the C-terminus and tobacco protoplasts, we have demonstrated that both can induce the hyperpolarization response. The results show that Nt-ERabp1 or the C-terminal peptide alone activates the auxin pathway from the outer face of the plasma membrane. (+info)
Foliar modifications induced by inhibition of polar transport of auxin.
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The effects of auxin polar transport inhibitors, 9-hydroxy-fluorene-9-carboxylic acid (HFCA); 2, 3, 5-triiodobenzoic acid (TIBA) and trans-cinnamic acid (CA) on leaf pattern formation were investigated with shoots formed from cultured leaf explants of tobacco and cultured pedicel explants of Orychophragmus violaceus, and the seedlings of tobacco and Brassica chinensis. Although the effective concentration varies with the inhibitors used, all of the inhibitors induced the formation of trumpet-shaped and/or fused leaves. The frequency of trumpet-shaped leaf formation was related to the concentration of inhibitors in the medium. Histological observation of tobacco seedlings showed that there was only one main vascular bundle and several minor vascular bundles in normal leaves of the control, but there were several vascular bundles of more or less the same size in the trumpet-shaped leaves of treated ones. These results indicated that auxin polar transport played an important role on bilateral symmetry of leaf growth. (+info)
Induction of ascorbate peroxidase by ethylene and hydrogen peroxide during growth of cultured soybean cells.
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In cultured soybean cells, a transient ethylene burst in the pre-stationary phase was followed by an induction of ascorbate peroxidase (AsPOX) in the stationary phase. Treatment of cells with the ethylene antagonist, silver thiosulfate (STS), resulted in the suppression of enzyme activity. Application of the ethylene releasing agent 2-chloroethylphosphonic acid (CEPA) in the medium led to an increased enzyme activity when treated in the pre-stationary phase. On the contrary, a remarkable inhibitory effect on enzyme activity was elicited by 1,3-dimethyl-2-thiourea (DMTU), trapping the hydrogen peroxide generated when treated in the stationary phase. Likewise, a steady level of AsPOX transcript was reduced by STS treatment. Furthermore, its effect appeared to be more rapid and prominent during the pre-stationary phase. It is suggested that the induction of AsPOX in cultured soybean cells during the stationary phase could result, at least in part, by the hydrogen peroxide generated as a result of preceding ethylene production. (+info)
Gene for a protein capable of enhancing lateral root formation.
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Analysis of genes preferentially expressed in hairy roots caused by infection with Agrobacterium rhizogenes has provided insights into the regulation of lateral root formation. A hairy root preferential cDNA, HR7, has been cloned from hairy roots of Hyoscyamus niger. HR7 encodes a novel protein partially homologous to a metallocarboxypeptidase inhibitor and is expressed exclusively in the primordium and base of lateral roots in hairy roots. Overexpression of HR7 in transgenic roots of H. niger dramatically enhances the frequency of lateral root formation. The results of this study indicate that expression of HR7 plays a critical role in initiating lateral root formation. (+info)