BACKGROUND: Plants rely on concentration gradients of the native auxin, indole-3-acetic acid (IAA), to modulate plant growth and development. Both metabolic and transport processes participate in the dynamic regulation of IAA homeostasis. Free IAA levels can be reduced by inactivation mechanisms, such as conjugation and degradation. IAA can be conjugated via ester linkage to glucose, or via amide linkage to amino acids, and degraded via oxidation. Members of the UDP glucosyl transferase (UGT) family catalyze the conversion of IAA to indole-3-acetyl-1-glucosyl ester (IAGlc); by contrast, IAA is irreversibly converted to indole-3-acetyl-l-aspartic acid (IAAsp) and indole-3-acetyl glutamic acid (IAGlu) by Group II of the GRETCHEN HAGEN3 (GH3) family of acyl amido synthetases. Dioxygenase for auxin oxidation (DAO) irreversibly oxidizes IAA to oxindole-3-acetic acid (oxIAA) and, in turn, oxIAA can be further glucosylated to oxindole-3-acetyl-1-glucosyl ester (oxIAGlc) by UGTs. These metabolic pathways have been identified based on mutant analyses, in vitro activity measurements, and in planta feeding assays. In vitro assays for studying protein activity are based on producing Arabidopsis enzymes in a recombinant form in bacteria or yeast followed by recombinant protein purification. However, the need to extract and purify the recombinant proteins represents a major obstacle when performing in vitro assays. RESULTS: In this work we report a rapid, reproducible and cheap method to screen the enzymatic activity of recombinant proteins that are known to inactivate IAA. The enzymatic reactions are carried out directly in bacteria that produce the recombinant protein. The enzymatic products can be measured by direct injection of a small supernatant fraction from the bacterial culture on ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UHPLC-ESI-MS/MS). Experimental procedures were optimized for testing the activity of different classes of IAA-modifying enzymes without the need to purify recombinant protein. CONCLUSIONS: This new method represents an alternative to existing in vitro assays. It can be applied to the analysis of IAA metabolites that are produced upon supplementation of substrate to engineered bacterial cultures and can be used for a rapid screening of orthologous candidate genes from non-model species.
- Klíčová slova
- Arabidopsis, Conjugation, Degradation, Enzyme assay, IAA metabolism, UHPLC–ESI-MS/MS,
- 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.
- Klíčová slova
- Arabidopsis thaliana, IAA degradation, dioxygenase, oxidase, root hair elongation,
- 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
- Názvy látek
- dioxygenasy MeSH
- indoleacetic acid MeSH Prohlížeč
- kyseliny indoloctové MeSH
- messenger RNA MeSH
- proteiny huseníčku MeSH
- zelené fluorescenční proteiny MeSH
The in vitro shoot propagation of Cannabis sativa L. is an emerging research area for large-scale plant material production. However, how in vitro conditions influence the genetic stability of maintained material, as well as whether changes in the concentration and composition of secondary metabolites can be expected are aspects that need to be better understood. These features are essential for the standardised production of medicinal cannabis. This work aimed to find out whether the presence of the auxin antagonist α-(2-oxo-2-phenylethyl)-1H-indole-3-acetic acid (PEO-IAA) in the culture media influenced the relative gene expression (RGE) of the genes of interest (OAC, CBCA, CBDA, THCA) and the concentrations of studied cannabinoids (CBCA, CBDA, CBC, ∆9-THCA, and ∆9-THC). Two C. sativa cultivars, 'USO-31' and 'Tatanka Pure CBD', were cultivated by in vitro conditions with PEO-IAA presence and then analysed. The RT-qPCR results indicated that even though some changes in the RGE profiles could be observed, no differences were statistically significant compared with the control variant. The results of the phytochemical analyses demonstrate that although there were some differences from the control variant, only the cultivar 'Tatanka Pure CBD' showed a statistically significant increase (at a statistical significance level α = 0.05) in the concentration of the cannabinoid CBDA. In conclusion, it would appear that using PEO-IAA in the culture medium is a suitable approach to improve in vitro cannabis multiplication.
- Klíčová slova
- CBC, CBD, Cannabis sativa, PEO-IAA, THC, auxin antagonist, cannabinoids, in vitro, qRT-PCR, shoot propagation,
- Publikační typ
- časopisecké články MeSH
N-Sulfonated IAA was discovered as a novel auxin metabolite in Urtica where it is biosynthesized de novo utilizing inorganic sulfate. It showed no auxin activity in DR5::GUS assay, implying possible inactivation/storage mechanism. A novel auxin derivative, N-sulfoindole-3-acetic acid (IAA-N-SO3H, SIAA), was discovered in stinging nettle (Urtica dioica) among 116 sulfonated metabolites putatively identified by a semi-targeted UHPLC-QqTOF-MS analysis of 23 plant/algae/fungi species. These sulfometabolites were detected based on the presence of a neutral loss of sulfur trioxide, as indicated by the m/z difference of 79.9568 Da in the MS2 spectra. The structure of newly discovered SIAA was confirmed by synthesizing its standard and comparing retention time, m/z and MS2 spectrum with those of SIAA found in Urtica. To study its natural occurrence, 73 species in total were further analyzed by UHPLC-QqTOF-MS or targeted UHPLC-MS/MS method with a limit of detection of 244 fmol/g dry weight. However, SIAA was only detected in Urtica at a concentration of 13.906 ± 9.603 nmol/g dry weight. Its concentration was > 30 times higher than that of indole-3-acetic acid (IAA), and the SIAA/IAA ratio was further increased under different light conditions, especially in continuous blue light. In addition to SIAA, structurally similar metabolites, N-sulfoindole-3-lactic acid, 4-(sulfooxy)phenyllactic acid and 4-(sulfooxy)phenylacetic acid, were detected in Urtica for the first time. SIAA was biosynthesized from inorganic sulfate in seedlings, as confirmed by the incorporation of exogenous 34S-ammonium sulfate (1 mM and 10 mM). SIAA exhibited no auxin activity, as demonstrated by both the Arabidopsis DR5::GUS assay and the Arabidopsis phenotype analysis. Sulfonation of IAA may therefore be a mechanism for IAA deactivation and/or storage in Urtica, similar to sulfonation of the jasmonates in Arabidopsis.
- Klíčová slova
- N-Sulfoindole-3-acetic acid, Indole-3-acetic acid, Mass spectrometry, Metabolomics, Phytohormone, Sulfonated,
- MeSH
- Arabidopsis metabolismus MeSH
- kyseliny indoloctové * metabolismus MeSH
- regulátory růstu rostlin metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- Urtica dioica metabolismus MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- indoleacetic acid MeSH Prohlížeč
- kyseliny indoloctové * MeSH
- regulátory růstu rostlin MeSH
Auxin phytohormones control most aspects of plant development through a complex and interconnected signaling network. In the presence of auxin, AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) transcriptional repressors are targeted for degradation by the SKP1-CULLIN1-F-BOX (SCF) ubiquitin-protein ligases containing TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB). CULLIN1-neddylation is required for SCFTIR1/AFB functionality, as exemplified by mutants deficient in the NEDD8-activating enzyme subunit AUXIN-RESISTANT 1 (AXR1). Here, we report a chemical biology screen that identifies small molecules requiring AXR1 to modulate plant development. We selected four molecules of interest, RubNeddin 1 to 4 (RN1 to -4), among which RN3 and RN4 trigger selective auxin responses at transcriptional, biochemical, and morphological levels. This selective activity is explained by their ability to consistently promote the interaction between TIR1 and a specific subset of AUX/IAA proteins, stimulating the degradation of particular AUX/IAA combinations. Finally, we performed a genetic screen using RN4, the RN with the greatest potential for dissecting auxin perception, which revealed that the chromatin remodeling ATPase BRAHMA is implicated in auxin-mediated apical hook development. These results demonstrate the power of selective auxin agonists to dissect auxin perception for plant developmental functions, as well as offering opportunities to discover new molecular players involved in auxin responses.
- Klíčová slova
- auxin, chemical biology, hormone perception, prohormone, selective agonist,
- MeSH
- Arabidopsis genetika metabolismus MeSH
- F-box proteiny metabolismus MeSH
- genetická transkripce účinky léků MeSH
- geneticky modifikované rostliny genetika MeSH
- kyseliny indoloctové metabolismus MeSH
- protein NEDD8 genetika MeSH
- proteinligasy komplexu SCF metabolismus MeSH
- proteiny huseníčku genetika metabolismus MeSH
- proteolýza * MeSH
- receptory buněčného povrchu metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- regulátory růstu rostlin genetika metabolismus MeSH
- semenáček metabolismus MeSH
- signální transdukce MeSH
- transkripční faktory metabolismus MeSH
- vývoj rostlin genetika fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- Názvy látek
- F-box proteiny MeSH
- indoleacetic acid MeSH Prohlížeč
- kyseliny indoloctové MeSH
- protein NEDD8 MeSH
- proteinligasy komplexu SCF MeSH
- proteiny huseníčku MeSH
- receptory buněčného povrchu MeSH
- regulátory růstu rostlin MeSH
- transkripční faktory MeSH
Together with auxin transport, auxin metabolism is a key determinant of auxin signaling output by plant cells. Enzymatic machinery involved in auxin metabolism is subject to regulation based on numerous inputs, including the concentration of auxin itself. Therefore, experiments characterizing altered auxin availability and subsequent changes in auxin metabolism could elucidate the function and regulatory role of individual elements in the auxin metabolic machinery. Here, we studied auxin metabolism in auxin-dependent tobacco BY-2 cells. We revealed that the concentration of N-(2-oxindole-3-acetyl)-l-aspartic acid (oxIAA-Asp), the most abundant auxin metabolite produced in the control culture, dramatically decreased in auxin-starved BY-2 cells. Analysis of the transcriptome and proteome in auxin-starved cells uncovered significant downregulation of all tobacco (Nicotiana tabacum) homologs of Arabidopsis (Arabidopsis thaliana) DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1), at both transcript and protein levels. Auxin metabolism profiling in BY-2 mutants carrying either siRNA-silenced or CRISPR-Cas9-mutated NtDAO1, as well as in dao1-1 Arabidopsis plants, showed not only the expected lower levels of oxIAA, but also significantly lower abundance of oxIAA-Asp. Finally, ability of DAO1 to oxidize IAA-Asp was confirmed by an enzyme assay in AtDAO1-producing bacterial culture. Our results thus represent direct evidence of DAO1 activity on IAA amino acid conjugates.
Root-hair growth and development regulated by soil microbes is associated with auxin. In this background, we hypothesized that mycorrhizal fungal inoculation induces greater root-hair growth through stimulated auxin synthesis and transport under water stress conditions. Trifoliate orange (Poncirus trifoliata) was inoculated with an arbuscular mycorrhizal (AM) fungus (Funneliformis mosseae) under well-watered (WW) and drought stress (DS) for 9 weeks. Compared with non-AM seedlings, AM seedlings displayed significantly higher density, length, and diameter of root hairs and root indoleacetic acid (IAA) level, whereas lower total root IAA efflux, regardless of soil moisture status. Root PtYUC3 and PtYUC8 involved in IAA biosynthesis were up-regulated by mycorrhization under WW and DS, whereas AM-modulated expression in PtTAA1, PtTAR2, PtYUC4, and PtYUC6 depended on status of soil moisture. Mycorrhizal inoculation down-regulated the transcript level of root auxin efflux carriers like PtPIN1 and PtPIN3, whereas significantly up-regulated the expression of root auxin-species influx carriers like PtABCB19 and PtLAX2 under DS. These results indicated that AMF-stimulated greater root-hair growth of trifoliate orange under DS that is independent on AMF species is related with mycorrhiza-modulated auxin synthesis and transport, which benefits the host plant to enhance drought tolerance.
- MeSH
- biologický transport MeSH
- fyziologický stres * MeSH
- Glomeromycota růst a vývoj fyziologie MeSH
- kyseliny indoloctové metabolismus MeSH
- messenger RNA genetika metabolismus MeSH
- mykorhiza růst a vývoj fyziologie MeSH
- období sucha * MeSH
- počet mikrobiálních kolonií MeSH
- Poncirus genetika růst a vývoj mikrobiologie fyziologie MeSH
- regulace genové exprese u rostlin MeSH
- rostlinné geny MeSH
- rostlinné proteiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- indoleacetic acid MeSH Prohlížeč
- kyseliny indoloctové MeSH
- messenger RNA MeSH
- rostlinné proteiny MeSH
Peptides derived from non-functional precursors play important roles in various developmental processes, but also in (a)biotic stress signaling. Our (phospho)proteome-wide analyses of C-TERMINALLY ENCODED PEPTIDE 5 (CEP5)-mediated changes revealed an impact on abiotic stress-related processes. Drought has a dramatic impact on plant growth, development and reproduction, and the plant hormone auxin plays a role in drought responses. Our genetic, physiological, biochemical, and pharmacological results demonstrated that CEP5-mediated signaling is relevant for osmotic and drought stress tolerance in Arabidopsis, and that CEP5 specifically counteracts auxin effects. Specifically, we found that CEP5 signaling stabilizes AUX/IAA transcriptional repressors, suggesting the existence of a novel peptide-dependent control mechanism that tunes auxin signaling. These observations align with the recently described role of AUX/IAAs in stress tolerance and provide a novel role for CEP5 in osmotic and drought stress tolerance.
- Klíčová slova
- Arabidopsis, Plant biology, developmental biology, hormones, label-free quantification, mass spectrometry, phosphoproteome, protein degradation, signal transduction, stress response,
- MeSH
- Arabidopsis genetika metabolismus fyziologie MeSH
- biologický transport genetika MeSH
- fosfoproteiny metabolismus MeSH
- fyziologická adaptace * genetika MeSH
- fyziologický stres * genetika MeSH
- genetická transkripce MeSH
- kyseliny indoloctové metabolismus MeSH
- období sucha MeSH
- osmóza MeSH
- peptidy metabolismus MeSH
- proteasomový endopeptidasový komplex metabolismus MeSH
- proteiny huseníčku metabolismus MeSH
- proteom metabolismus MeSH
- proteomika * MeSH
- regulace genové exprese u rostlin MeSH
- semenáček růst a vývoj MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- Názvy látek
- CEP5 protein, Arabidopsis MeSH Prohlížeč
- fosfoproteiny MeSH
- kyseliny indoloctové MeSH
- peptidy MeSH
- proteasomový endopeptidasový komplex MeSH
- proteiny huseníčku MeSH
- proteom 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
- Názvy látek
- F-box proteiny MeSH
- herbicidy MeSH
- kyselina 2,4-dichlorfenoxyoctová MeSH
- kyseliny indoloctové MeSH
- proteiny huseníčku MeSH
- receptory buněčného povrchu MeSH
- regulátory růstu rostlin MeSH
- TIR1 protein, Arabidopsis MeSH Prohlížeč
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.
- Klíčová slova
- Arabidopsis, AuxIAA, TIR1/AFB, adventitious roots, jasmonate,
- 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
- Názvy látek
- AFB2 protein, Arabidopsis MeSH Prohlížeč
- F-box proteiny MeSH
- indoleacetic acid MeSH Prohlížeč
- kyseliny indoloctové MeSH
- proteiny huseníčku MeSH
- receptory buněčného povrchu MeSH
- TIR1 protein, Arabidopsis MeSH Prohlížeč
