auxin conjugates Dotaz Zobrazit nápovědu
Bacterial metabolism of phytohormones includes several processes such as biosynthesis, catabolism, conjugation, hydrolysis and homeostatic regulation. However, only biosynthesis and occasionally catabolism are studied in depth in microorganisms. In this work, we evaluated and reconsidered IAA metabolism in Bradyrhizobiumjaponicum E109, one of the most widely used strains for soybean inoculation around the world. The genomic analysis of the strain showed the presence of several genes responsible for IAA biosynthesis, mainly via indole-3-acetonitrile (IAN), indole-3-acetamide (IAM) and tryptamine (TAM) pathways. However; in vitro experiments showed that IAA is not accumulated in the culture medium in significant amounts. On the contrary, a strong degradation activity was observed after exogenous addition of 0.1 mM of IAA, IBA or NAA to the medium. B. japonicum E109 was not able to grow in culture medium containing IAA as a sole carbon source. In YEM medium, the bacteria degraded IAA and hydrolyzed amino acid auxin conjugates with alanine (IAAla), phenylalanine (IAPhe), and leucine (IAPhe), releasing IAA which was quickly degraded. Finally, the presence of exogenous IAA induced physiological changes in the bacteria such as increased biomass and exopolysaccharide production, as well as infection effectiveness and symbiotic behavior in soybean plants.
- MeSH
- alanin metabolismus MeSH
- bakteriální polysacharidy biosyntéza MeSH
- Bradyrhizobium genetika metabolismus MeSH
- fenylalanin metabolismus MeSH
- Glycine max mikrobiologie MeSH
- kyseliny indoloctové metabolismus MeSH
- leucin metabolismus MeSH
- semena rostlinná mikrobiologie MeSH
- symbióza fyziologie MeSH
- tvorba kořenových hlízek fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
Plant development mediated by the phytohormone auxin depends on tightly controlled cellular auxin levels at its target tissue that are largely established by intercellular and intracellular auxin transport mediated by PIN auxin transporters. Among the eight members of the Arabidopsis PIN family, PIN6 is the least characterized candidate. In this study we generated functional, fluorescent protein-tagged PIN6 proteins and performed comprehensive analysis of their subcellular localization and also performed a detailed functional characterization of PIN6 and its developmental roles. The localization study of PIN6 revealed a dual localization at the plasma membrane (PM) and endoplasmic reticulum (ER). Transport and metabolic profiling assays in cultured cells and Arabidopsis strongly suggest that PIN6 mediates both auxin transport across the PM and intracellular auxin homeostasis, including the regulation of free auxin and auxin conjugates levels. As evidenced by the loss- and gain-of-function analysis, the complex function of PIN6 in auxin transport and homeostasis is required for auxin distribution during lateral and adventitious root organogenesis and for progression of these developmental processes. These results illustrate a unique position of PIN6 within the family of PIN auxin transporters and further add complexity to the developmentally crucial process of auxin transport.
- MeSH
- Arabidopsis genetika růst a vývoj metabolismus MeSH
- buněčná membrána metabolismus MeSH
- endoplazmatické retikulum metabolismus MeSH
- fylogeneze MeSH
- geneticky modifikované rostliny MeSH
- homeostáza MeSH
- kořeny rostlin růst a vývoj metabolismus MeSH
- kyseliny indoloctové metabolismus MeSH
- membránové transportní proteiny genetika metabolismus MeSH
- molekulární evoluce MeSH
- proteiny huseníčku genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The plant hormone auxin is a key player in the regulation of plant growth and development. Despite numerous studies devoted to understanding its role in a wide spectrum of physiological processes, full appreciation of its function is linked to a comprehensive determination of its spatio-temporal distribution, which plays a crucial role in its mode of action. Conjugation of fluorescent tracers to plant hormones enables sensitive and specific visualization of their subcellular and tissue-specific localization and transport in planta, which represents a powerful tool for plant physiology. However, to date, only a few fluorescently labeled auxins have been developed. We report the synthesis of four novel fluorescently labeled derivatives of indole-3-acetic acid (IAA) in the form of a conjugate with a nitrobenzoxadiazole (NBD) fluorophore together with validation of their biological activity. These compounds, unlike other previously reported auxins fluorescently labeled at N1 position (nitrogen of the indole ring), do not possess auxin activity but rather show dose-dependent inhibition of auxin-induced effects, such as primary root growth inhibition, root hair growth and the auxin reporter DR5::GUS expression. Moreover, the study demonstrates the importance of the character of the linker and optimal choice of the labeling site in the preparation of fluorescently labeled auxins as important variables influencing their biological activity and fluorescent properties.
- MeSH
- Arabidopsis účinky léků genetika růst a vývoj MeSH
- fluorescenční barviva chemická syntéza chemie MeSH
- fluorescenční spektrometrie MeSH
- geneticky modifikované rostliny MeSH
- kořeny rostlin účinky léků růst a vývoj MeSH
- kyseliny indoloctové antagonisté a inhibitory chemie farmakologie MeSH
- molekulární struktura MeSH
- regulátory růstu rostlin antagonisté a inhibitory chemie farmakologie MeSH
- spektrofotometrie MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND AND AIMS: The typical rootless linear shoots of aquatic carnivorous plants exhibit clear, steep polarity associated with very rapid apical shoot growth. The aim of this study was to determine how auxin and cytokinin contents are related to polarity and shoot growth in such plants. METHODS: The main auxin and cytokinin metabolites in separated shoot segments and turions of two carnivorous plants, Aldrovanda vesiculosa and Utricularia australis, were analysed using ultra-high-performance liquid chromatography coupled with triple quad mass spectrometry. KEY RESULTS: In both species, only isoprenoid cytokinins were identified. Zeatin cytokinins predominated in the apical parts, with their concentrations decreasing basipetally, and the trans isomer predominated in A. vesiculosa whereas the cis form was more abundant in U australis. Isopentenyladenine-type cytokinins, in contrast, increased basipetally. Conjugated cytokinin metabolites, the O-glucosides, were present at high concentrations in A. vesiculosa but only in minute amounts in U. australis. N(9)-glucoside forms were detected only in U. australis, with isopentenyladenine-9-glucoside (iP9G) being most abundant. In addition to free indole-3-acetic acid (IAA), indole-3-acetamide (IAM), IAA-aspartate (IAAsp), IAA-glutamate (IAGlu) and IAA-glycine (IAGly) conjugates were identified. CONCLUSIONS: Both species show common trends in auxin and cytokinin levels, the apical localization of the cytokinin biosynthesis and basipetal change in the ratio of active cytokinins to auxin, in favour of auxin. However, our detailed study of cytokinin metabolic profiles also revealed that both species developed different regulatory mechanisms of active cytokinin content; on the level of their degradation, in U. australis, or in the biosynthesis itself, in the case of A. vesiculosa Results indicate that the rapid turnover of these signalling molecules along the shoots is essential for maintaining the dynamic balance between the rapid polar growth and development of the apical parts and senescence of the older, basal parts of the shoots.
Auxin (indole-3-acetic acid, IAA) plays fundamental roles as a signalling molecule during numerous plant growth and development processes. The formation of local auxin gradients and auxin maxima/minima, which is very important for these processes, is regulated by auxin metabolism (biosynthesis, degradation, and conjugation) as well as transport. When studying auxin metabolism pathways it is crucial to combine data obtained from genetic investigations with the identification and quantification of individual metabolites. Thus, to facilitate efforts to elucidate auxin metabolism and its roles in plants, we have developed a high-throughput method for simultaneously quantifying IAA and its key metabolites in minute samples (<10 mg FW) of Arabidopsis thaliana tissues by in-tip micro solid-phase extraction and fast LC-tandem MS. As a proof of concept, we applied the method to a collection of Arabidopsis mutant lines and identified lines with altered IAA metabolite profiles using multivariate data analysis. Finally, we explored the correlation between IAA metabolite profiles and IAA-related phenotypes. The developed rapid analysis of large numbers of samples (>100 samples d-1) is a valuable tool to screen for novel regulators of auxin metabolism and homeostasis among large collections of genotypes.
- MeSH
- Arabidopsis genetika metabolismus MeSH
- chromatografie kapalinová MeSH
- extrakce na pevné fázi MeSH
- kyseliny indoloctové metabolismus MeSH
- multivariační analýza MeSH
- mutace * MeSH
- rostlinné proteiny analýza MeSH
- tandemová hmotnostní spektrometrie MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The phenylpropanoid 3,4-(methylenedioxy)cinnamic acid (MDCA) is a plant-derived compound first extracted from roots of Asparagus officinalis and further characterized as an allelochemical. Later on, MDCA was identified as an efficient inhibitor of 4-COUMARATE-CoA LIGASE (4CL), a key enzyme of the general phenylpropanoid pathway. By blocking 4CL, MDCA affects the biosynthesis of many important metabolites, which might explain its phytotoxicity. To decipher the molecular basis of the allelochemical activity of MDCA, we evaluated the effect of this compound on Arabidopsis thaliana seedlings. Metabolic profiling revealed that MDCA is converted in planta into piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H), the enzyme directly upstream of 4CL. The inhibition of C4H was also reflected in the phenolic profile of MDCA-treated plants. Treatment of in vitro grown plants resulted in an inhibition of primary root growth and a proliferation of lateral and adventitious roots. These observed growth defects were not the consequence of lignin perturbation, but rather the result of disturbing auxin homeostasis. Based on DII-VENUS quantification and direct measurement of cellular auxin transport, we concluded that MDCA disturbs auxin gradients by interfering with auxin efflux. In addition, mass spectrometry was used to show that MDCA triggers auxin biosynthesis, conjugation, and catabolism. A similar shift in auxin homeostasis was found in the c4h mutant ref3-2, indicating that MDCA triggers a cross talk between the phenylpropanoid and auxin biosynthetic pathways independent from the observed auxin efflux inhibition. Altogether, our data provide, to our knowledge, a novel molecular explanation for the phytotoxic properties of MDCA.
- MeSH
- 4-monooxygenasa kyseliny skořicové antagonisté a inhibitory metabolismus MeSH
- Arabidopsis účinky léků genetika metabolismus MeSH
- benzoáty metabolismus farmakologie MeSH
- biosyntetické dráhy účinky léků MeSH
- cinnamáty chemie metabolismus farmakologie MeSH
- fenylpropionáty chemie metabolismus farmakologie MeSH
- geneticky modifikované rostliny MeSH
- hmotnostní spektrometrie MeSH
- homeostáza účinky léků MeSH
- koenzym A-ligasy antagonisté a inhibitory metabolismus MeSH
- konfokální mikroskopie MeSH
- kořeny rostlin účinky léků genetika metabolismus MeSH
- kyseliny indoloctové metabolismus MeSH
- lignin biosyntéza MeSH
- semenáček účinky léků genetika růst a vývoj metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Publikační typ
- časopisecké články MeSH
In Arabidopsis thaliana, canonical auxin-dependent gene regulation is mediated by 23 transcription factors from the AUXIN RESPONSE FACTOR (ARF) family that interact with auxin/indole acetic acid repressors (Aux/IAAs), which themselves form co-receptor complexes with one of six TRANSPORT INHIBITOR1/AUXIN-SIGNALLING F-BOX (TIR1/AFB) proteins. Different combinations of co-receptors drive specific sensing outputs, allowing auxin to control a myriad of processes. ARF6 and ARF8 are positive regulators of adventitious root initiation upstream of jasmonate, but the exact auxin co-receptor complexes controlling the transcriptional activity of these proteins has remained unknown. Here, using loss-of-function mutants we show that three Aux/IAA genes, IAA6, IAA9, and IAA17, act additively in the control of adventitious root (AR) initiation. These three IAA proteins interact with ARF6 and/or ARF8 and likely repress their activity in AR development. We show that TIR1 and AFB2 are positive regulators of AR formation and TIR1 plays a dual role in the control of jasmonic acid (JA) biosynthesis and conjugation, as several JA biosynthesis genes are up-regulated in the tir1-1 mutant. These results lead us to propose that in the presence of auxin, TIR1 and AFB2 form specific sensing complexes with IAA6, IAA9, and/or IAA17 to modulate JA homeostasis and control AR initiation.
- MeSH
- Arabidopsis cytologie genetika růst a vývoj metabolismus MeSH
- F-box proteiny metabolismus MeSH
- hypokotyl metabolismus MeSH
- kořeny rostlin růst a vývoj MeSH
- kyseliny indoloctové metabolismus MeSH
- proteiny huseníčku metabolismus MeSH
- receptory buněčného povrchu metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- signální transdukce * MeSH
- stabilita proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The herbicide 2,4-D exhibits an auxinic activity and therefore can be used as a synthetic and traceable analog to study auxin-related responses. Here we identified that not only exogenous 2,4-D but also its amide-linked metabolite 2,4-D-Glu displayed an inhibitory effect on plant growth via the TIR1/AFB auxin-mediated signaling pathway. To further investigate 2,4-D metabolite conversion, identity and activity, we have developed a novel purification procedure based on the combination of ion exchange and immuno-specific sorbents combined with a sensitive liquid chromatography-mass spectrometry method. In 2,4-D treated samples, 2,4-D-Glu and 2,4-D-Asp were detected at 100-fold lower concentrations compared to 2,4-D levels, showing that 2,4-D can be metabolized in the plant. Moreover, 2,4-D-Asp and 2,4-D-Glu were identified as reversible forms of 2,4-D homeostasis that can be converted to free 2,4-D. This work paves the way to new studies of auxin action in plant development.
- MeSH
- Arabidopsis účinky léků růst a vývoj metabolismus MeSH
- F-box proteiny genetika metabolismus MeSH
- herbicidy farmakologie MeSH
- homeostáza MeSH
- kořeny rostlin účinky léků růst a vývoj metabolismus MeSH
- kyselina 2,4-dichlorfenoxyoctová farmakologie MeSH
- kyseliny indoloctové metabolismus MeSH
- proteiny huseníčku genetika metabolismus MeSH
- receptory buněčného povrchu genetika metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- regulátory růstu rostlin farmakologie MeSH
- semenáček účinky léků růst a vývoj metabolismus MeSH
- signální transdukce účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
Auxin represents a key signal in plants, regulating almost every aspect of their growth and development. Major breakthroughs have been made dissecting the molecular basis of auxin transport, perception, and response. In contrast, how plants control the metabolism and homeostasis of the major form of auxin in plants, indole-3-acetic acid (IAA), remains unclear. In this paper, we initially describe the function of the Arabidopsis thaliana gene DIOXYGENASE FOR AUXIN OXIDATION 1 (AtDAO1). Transcriptional and translational reporter lines revealed that AtDAO1 encodes a highly root-expressed, cytoplasmically localized IAA oxidase. Stable isotope-labeled IAA feeding studies of loss and gain of function AtDAO1 lines showed that this oxidase represents the major regulator of auxin degradation to 2-oxoindole-3-acetic acid (oxIAA) in Arabidopsis Surprisingly, AtDAO1 loss and gain of function lines exhibited relatively subtle auxin-related phenotypes, such as altered root hair length. Metabolite profiling of mutant lines revealed that disrupting AtDAO1 regulation resulted in major changes in steady-state levels of oxIAA and IAA conjugates but not IAA. Hence, IAA conjugation and catabolism seem to regulate auxin levels in Arabidopsis in a highly redundant manner. We observed that transcripts of AtDOA1 IAA oxidase and GH3 IAA-conjugating enzymes are auxin-inducible, providing a molecular basis for their observed functional redundancy. We conclude that the AtDAO1 gene plays a key role regulating auxin homeostasis in Arabidopsis, acting in concert with GH3 genes, to maintain auxin concentration at optimal levels for plant growth and development.
- MeSH
- Arabidopsis enzymologie genetika MeSH
- biologické modely MeSH
- dioxygenasy metabolismus MeSH
- fenotyp MeSH
- fylogeneze MeSH
- homeostáza * MeSH
- kořeny rostlin metabolismus MeSH
- kyseliny indoloctové metabolismus MeSH
- messenger RNA genetika metabolismus MeSH
- metabolomika MeSH
- mutace genetika MeSH
- oxidace-redukce MeSH
- promotorové oblasti (genetika) genetika MeSH
- proteiny huseníčku chemie genetika metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- rostlinné geny * MeSH
- sekvence aminokyselin MeSH
- semenáček metabolismus MeSH
- výhonky rostlin metabolismus MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
