Auxins are hormones that regulate growth and development in plants. Besides plants, various microorganisms also produce auxins. Here we investigate whether and how the phytopathogenic fungus Leptosphaeria maculans biosynthesizes auxins. We characterized the auxin profile of in vitro grown L. maculans. The culture was further supplied with the auxin biosynthetic-precursors tryptophan and tryptamine and gene expression and phytohormone content was analyzed. L. maculans in vitro produced IAA (indole-3-acetic acid) as the predominant auxin metabolite. IAA production could be further stimulated by supplying precursors. Expression of indole-3-pyruvate decarboxylase LmIPDC2, tryptophan aminotransferase LmTAM1 and nitrilase LmNIT1 genes was mainly upregulated after adding tryptophan and correlated with IAA production, suggesting that these genes are the key components of auxin biosynthesis in L. maculans. Tryptamine acted as a potent inducer of IAA production, though a pathway independent of LmIPDC2/LmTAM1 may be involved. Despite L. maculans being a rich source of bioactive IAA, the auxin metabolic profile of host plant Brassica napus was not altered upon infection. Exogenous IAA inhibited the growth of L. maculans in vitro when supplied in high concentration. Altogether, we showed that L. maculans is capable of IAA production and we have identified biosynthetic genes that were responsive to tryptophan treatment.

• Klíčová slova
• Dothideomycetes, Hormone, Indole-3-pyruvate decarboxylase, Tryptamine, Tryptophan, Tryptophan aminotransferase,
• MeSH
• aminohydrolasy genetika   MeSH
• biosyntetické dráhy MeSH
• Brassica napus mikrobiologie   MeSH
• fylogeneze MeSH
• genetická transkripce MeSH
• houby klasifikace   genetika   metabolismus   MeSH
• karboxylyasy genetika   metabolismus   MeSH
• kyseliny indoloctové metabolismus   farmakologie   MeSH
• Leptosphaeria enzymologie   genetika   růst a vývoj   metabolismus   MeSH
• regulace genové exprese u hub MeSH
• regulátory růstu rostlin metabolismus   MeSH
• tryptaminy metabolismus   farmakologie   MeSH
• tryptofan metabolismus   farmakologie   MeSH
• tryptofantransaminasa genetika   metabolismus   MeSH
• upregulace MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminohydrolasy MeSH
• indoleacetic acid MeSH Prohlížeč
• indolepyruvate decarboxylase MeSH Prohlížeč
• karboxylyasy MeSH
• kyseliny indoloctové MeSH
• nitrilase MeSH Prohlížeč
• regulátory růstu rostlin MeSH
• tryptamine MeSH Prohlížeč
• tryptaminy MeSH
• tryptofan MeSH
• tryptofantransaminasa MeSH

Identification of mutants with impairments in auxin biosynthesis and dynamics by forward genetic screening is hindered by the complexity, redundancy and necessity of the pathways involved. Furthermore, although a few auxin-deficient mutants have been recently identified by screening for altered responses to shade, ethylene, N-1-naphthylphthalamic acid (NPA) or cytokinin (CK), there is still a lack of robust markers for systematically isolating such mutants. We hypothesized that a potentially suitable phenotypic marker is root curling induced by CK, as observed in the auxin biosynthesis mutant CK-induced root curling 1 / tryptophan aminotransferase of Arabidopsis 1 (ckrc1/taa1). Phenotypic observations, genetic analyses and biochemical complementation tests of Arabidopsis seedlings displaying the trait in large-scale genetic screens showed that it can facilitate isolation of mutants with perturbations in auxin biosynthesis, transport and signaling. However, unlike transport/signaling mutants, the curled (or wavy) root phenotypes of auxin-deficient mutants were significantly induced by CKs and could be rescued by exogenous auxins. Mutants allelic to several known auxin biosynthesis mutants were re-isolated, but several new classes of auxin-deficient mutants were also isolated. The findings show that CK-induced root curling provides an effective marker for discovering genes involved in auxin biosynthesis or homeostasis.

• MeSH
• Arabidopsis enzymologie   genetika   MeSH
• biologický transport účinky léků   MeSH
• cytokininy metabolismus   MeSH
• fenotyp MeSH
• kořeny rostlin enzymologie   genetika   růst a vývoj   MeSH
• kyseliny indoloctové metabolismus   farmakologie   MeSH
• mutace MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• regulátory růstu rostlin biosyntéza   MeSH
• semenáček účinky léků   růst a vývoj   metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• tryptofantransaminasa genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokininy MeSH
• indoleacetic acid MeSH Prohlížeč
• kyseliny indoloctové MeSH
• proteiny huseníčku MeSH
• regulátory růstu rostlin MeSH
• tryptofantransaminasa MeSH

Transamination of tryptophan belongs to minor pathways of amino acid metabolism. The present paper describes conditions for application of dinitrophenylhydrazine method, originally prepared for alanine aminortansferase and aspartate aminotransferase assay, to the measurement of tryptophan transamination catalysed by any of the enzymes mentioned above. The method was tested using purified pig heart AST. While the free enzyme showed a characteristic absorption profile with the maxima at 360 and 430 nm, the course of transamination of tryptophan was confirmed by the measurement of UV-VIS spectral changes of the coenzyme in the active site of the enzyme in the presence of the amino acid substrate only, when tryptophan caused a shift of the peak from 360 nm to 330 nm due to a change of the pyridoxal form to the pyridoxamine form (= the first step of ping-pong transaminating reaction). A general limitation of dinitrophenylhydrazine method is the interference of hydrazones formed from the coenzyme pyridoxal-5'-phosphate and from the oxo- substrate 2-oxoglutarate, showing the absorption maxima at 492 nm and 388 nm, respectively with the hydrazones formed by the oxo- products (pyruvate and/or oxaloacetate in the case of ALT/AST, the absorption maxima at 443 nm in our measurements). In the case of tryptophan transamination, indolepyruvate as the oxo- product of a catalysed reaction forms dinitrophenylhydrazone, which has, besides a maximum at 435 nm, a distinct peak at 542 nm, convenient for the product concentration measurement. This is favourable for resolution from other (interfering) hydrazones. Suitable conditions for tryptophan transamination in tissue and enzyme preparations were found. Reaching optimal conditions for tryptophan transamination measurements in vitro is generally limited by low solubility of the amino acid in water solutions: With AST preparation, the velocity of catalysed reaction at 5-50 x 10(-3) M tryptophan concentration was of 1st order to the amino acid substrate. Km for tryptophan was found > or = 2 x 10(-1) M. Therefore the enzyme activity measurement at two different tryptophan concentrations is recommended for unknown samples. Tryptophan transamination by purified pig AST was compared with that catalysed by preparations obtained from mammalian tissues.

• MeSH
• aspartátaminotransferasy metabolismus   MeSH
• hydraziny metabolismus   MeSH
• játra enzymologie   MeSH
• kinetika MeSH
• krysa rodu Rattus MeSH
• mozek enzymologie   MeSH
• myokard enzymologie   MeSH
• potkani Wistar MeSH
• substrátová specifita MeSH
• Sus scrofa MeSH
• techniky in vitro MeSH
• tenké střevo enzymologie   MeSH
• transaminasy metabolismus   MeSH
• tryptofan metabolismus   MeSH
• tryptofantransaminasa MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aspartátaminotransferasy MeSH
• dinitrophenylhydrazine MeSH Prohlížeč
• hydraziny MeSH
• transaminasy MeSH
• tryptofan MeSH
• tryptofantransaminasa MeSH