Conversion of Unstable Compounds Can Contribute to the Auxin Pool during Sample Preparation

Gélinas-Marion, Ariane; Nichols, David S.; Ross, John J.

doi:10.1104/pp.20.00251

Cited by 7 publications

(7 citation statements)

References 15 publications

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“…While Bs3 and YUCCAs seem to differ in their metabolic product, it would seem possible that both enzymes use IPA as a metabolic substrate. Unfortunately, IPA is a rather unstable metabolite that spontaneously converts into IAA in vitro [ 29 ]. In consequence, quantification of IPA consumption by recombinant Bs3 protein in vitro proves to be rather challenging.…”

Section: Discussionmentioning

confidence: 99%

The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H2O2 in vitro

Krönauer

Lahaye

2021

PLoS ONE

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The pepper resistance gene Bs3 triggers a hypersensitive response (HR) upon transcriptional activation by the corresponding effector protein AvrBs3 from the bacterial pathogen Xanthomonas. Expression of Bs3 in yeast inhibited proliferation, demonstrating that Bs3 function is not restricted to the plant kingdom. The Bs3 sequence shows striking similarity to flavin monooxygenases (FMOs), an FAD- and NADPH-containing enzyme class that is known for the oxygenation of a wide range of substrates and their potential to produce H2O2. Since H2O2 is a hallmark metabolite in plant immunity, we analyzed the role of H2O2 during Bs3 HR. We purified recombinant Bs3 protein from E. coli and confirmed the FMO function of Bs3 with FAD binding and NADPH oxidase activity in vitro. Translational fusion of Bs3 to the redox reporter roGFP2 indicated that the Bs3-dependent HR induces an increase of the intracellular oxidation state in planta. To test if the NADPH oxidation and putative H2O2 production of Bs3 is sufficient to induce HR, we adapted previous studies which have uncovered mutations in the NADPH binding site of FMOs that result in higher NADPH oxidase activity. In vitro studies demonstrated that recombinant Bs3S211A protein has twofold higher NADPH oxidase activity than wildtype Bs3. Translational fusions to roGFP2 showed that Bs3S211A also increased the intracellular oxidation state in planta. Interestingly, while the mutant derivative Bs3S211A had an increase in NADPH oxidase capacity, it did not trigger HR in planta, ultimately revealing that H2O2 produced by Bs3 on its own is not sufficient to trigger HR.

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Section: Discussionmentioning

confidence: 99%

The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H2O2 in vitro

Krönauer

Lahaye

2021

PLoS ONE

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“…Each independent experiment used at least three biological replicates. Sample handling times were kept as short as possible to prevent distortion of the IAA pool through the autocatalytic decay of precursor molecules ( Gélinas-Marion et al , 2020 ). The plant material was transferred into 2 ml screw-cap tubes filled with ceramic beads (MagNA Lyser Green Beads; Roche Diagnostics) and endogenous IAA and IAM was extracted into 1 ml of cold potassium phosphate buffer (50 mM, pH 7.0) containing 1% diethyldithiocarbamic acid sodium salt and 50 pmol of the internal standards [ 2 H 5 ]IAA and [ 2 H 5 , 15 N]IAM (OlchemIm).…”

Section: Methodsmentioning

confidence: 99%

Endogenous indole-3-acetamide levels contribute to the crosstalk between auxin and abscisic acid, and trigger plant stress responses in Arabidopsis

Pérez‐Alonso

Ortiz‐García

Moya-Cuevas

et al. 2020

Journal of Experimental Botany

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The evolutionary success of plants relies to a large extent on their extraordinary ability to adapt to changes in their environment. These adaptations require that plants balance their growth with their stress responses. Plant hormones are crucial mediators orchestrating the underlying adaptive processes. However, whether and how the growth-related hormone auxin and the stress-related hormones jasmonic acid (JA), salicylic acid, and abscisic acid (ABA) are coordinated remains largely elusive. Here, we analyze the physiological role of AMIDASE 1 (AMI1) in plant growth and its possible connection to plant adaptations to abiotic stresses. AMI1 contributes to cellular auxin homeostasis by catalyzing the conversion of indole-acetamide into the major plant auxin indole-3-acetic acid. Functional impairment of AMI1 increases the plants’ stress status rendering mutant plants more susceptible to abiotic stresses. Transcriptomic analysis of ami1 mutants disclosed the reprogramming of a considerable number of stress-related genes, including JA and ABA biosynthesis genes. The ami1 mutants exhibit only moderately repressed growth, but an enhanced ABA accumulation, which suggests a role for AMI1 in the crosstalk between auxin and ABA. Altogether, our results suggest that AMI1 is involved in coordinating the trade-off between plant growth and stress responses, balancing auxin with ABA homeostasis.

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“…In the case of Aux analysis, biosynthetic precursors exhibit limited stability and undergo changes during extraction, leading to overestimation of the IAA content. Artifact formation is pronounced when using aqueous buffers for extraction or under pH extremes, leading to hydrolysis of conjugated forms or chemical modifications [ 115 – 117 ]. The use of aqueous buffers for Aux extraction also raises concerns about possible enzymatic activity [ 118 ].…”

Section: Analysis Of Plant Hormonesmentioning

confidence: 99%

“…A mixture of IPA:imidazole buffers is a suitable option for Aux extraction [ 116 ]. Furthermore, overestimation of IAA due to the instability of Aux intermediates can be handled by either adding antioxidants [ 115 , 119 ] or derivatization, e.g. using methoxamine [ 120 ] or cysteamine [ 121 , 122 ].…”

Section: Analysis Of Plant Hormonesmentioning

confidence: 99%

Can plant hormonomics be built on simple analysis? A review

Vrobel,

Tarkowski

2023

Plant Methods

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The field of plant hormonomics focuses on the qualitative and quantitative analysis of the hormone complement in plant samples, akin to other omics sciences. Plant hormones, alongside primary and secondary metabolites, govern vital processes throughout a plant's lifecycle. While active hormones have received significant attention, studying all related compounds provides valuable insights into internal processes. Conventional single-class plant hormone analysis employs thorough sample purification, short analysis and triple quadrupole tandem mass spectrometry. Conversely, comprehensive hormonomics analysis necessitates minimal purification, robust and efficient separation and better-performing mass spectrometry instruments. This review summarizes the current status of plant hormone analysis methods, focusing on sample preparation, advances in chromatographic separation and mass spectrometric detection, including a discussion on internal standard selection and the potential of derivatization. Moreover, current approaches for assessing the spatiotemporal distribution are evaluated. The review touches on the legitimacy of the term plant hormonomics by exploring the current status of methods and outlining possible future trends.

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Conversion of Unstable Compounds Can Contribute to the Auxin Pool during Sample Preparation

Cited by 7 publications

References 15 publications

The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H2O2 in vitro

The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H2O2 in vitro

Endogenous indole-3-acetamide levels contribute to the crosstalk between auxin and abscisic acid, and trigger plant stress responses in Arabidopsis

Can plant hormonomics be built on simple analysis? A review

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