What Has Been Seen Cannot Be Unseen—Detecting Auxin In Vivo

Pařízková, Barbora; Pernisová, Markéta; Novák, Ondřej

doi:10.3390/ijms18122736

Cited by 29 publications

(15 citation statements)

References 216 publications

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“…Analytical methods for quantitation of auxins and CKs have become increasingly sensitive and capable of discriminating not only the free hormones, but also many of their precursors, metabolites, and catabolites [ 7 , 8 ]. Nevertheless, at the subcellular level, organelle-specific phytohormone profiling is challenging and many factors need to be optimized, such as (i) leakage during isolation; (ii) purity of isolated compartments; and (iii) dynamic metabolic changes during isolation.…”

Section: Organelle-specific Phytohormone Profilingmentioning

confidence: 99%

“…Mass spectrometry imaging and living single-cell mass spectrometry analysis could soon provide powerful tools for studying hormone distribution, even though they are still limited for hormone profiling [ 8 ]. Cell-specific sorting has been employed to gain more accurate insight into auxin [ 33 , 185 ] and CK [ 186 ] distributions in Arabidopsis root tips, and flow cytometric techniques for the sorting of organelles may soon provide a better view on subcellular distributions.…”

Section: Future Perspectivesmentioning

confidence: 99%

See 1 more Smart Citation

Auxins and Cytokinins—The Role of Subcellular Organization on Homeostasis

Skalický

Kubeš

Napier

et al. 2018

IJMS

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Plant hormones are master regulators of plant growth and development. Better knowledge of their spatial signaling and homeostasis (transport and metabolism) on the lowest structural levels (cellular and subcellular) is therefore crucial to a better understanding of developmental processes in plants. Recent progress in phytohormone analysis at the cellular and subcellular levels has greatly improved the effectiveness of isolation protocols and the sensitivity of analytical methods. This review is mainly focused on homeostasis of two plant hormone groups, auxins and cytokinins. It will summarize and discuss their tissue- and cell-type specific distributions at the cellular and subcellular levels.

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Section: Organelle-specific Phytohormone Profilingmentioning

confidence: 99%

Section: Future Perspectivesmentioning

confidence: 99%

Auxins and Cytokinins—The Role of Subcellular Organization on Homeostasis

Skalický

Kubeš

Napier

et al. 2018

IJMS

Self Cite

View full text Add to dashboard Cite

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“…Auxin‐specific genetic reporters enable tissue‐specific localisation of auxin activity as a result of the expression of markers (e.g. β‐glucuronidase (GUS) or fluorescent proteins) fused to auxin‐sensitive promoters (Pařízková et al ., 2017). In addition, degradation‐based reporters have been constructed to provide sensitive and rapid responses to the presence of auxin at a cellular resolution.…”

Section: Introductionmentioning

confidence: 99%

New fluorescent auxin probes visualise tissue‐specific and subcellular distributions of auxin in Arabidopsis

Pařízková

Žukauskaitė

Vain³

et al. 2021

New Phytologist

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Summary In a world that will rely increasingly on efficient plant growth for sufficient food, it is important to learn about natural mechanisms of phytohormone action. In this work, the introduction of a fluorophore to an auxin molecule represents a sensitive and non‐invasive method to directly visualise auxin localisation with high spatiotemporal resolution. The state‐of‐the‐art multidisciplinary approaches of genetic and chemical biology analysis together with live cell imaging, liquid chromatography–mass spectrometry (LC‐MS) and surface plasmon resonance (SPR) methods were employed for the characterisation of auxin‐related biological activity, distribution and stability of the presented compounds in Arabidopsis thaliana. Despite partial metabolisation in vivo, these fluorescent auxins display an uneven and dynamic distribution leading to the formation of fluorescence maxima in tissues known to concentrate natural auxin, such as the concave side of the apical hook. Importantly, their distribution is altered in response to different exogenous stimuli in both roots and shoots. Moreover, we characterised the subcellular localisation of the fluorescent auxin analogues as being present in the endoplasmic reticulum and endosomes. Our work provides powerful tools to visualise auxin distribution within different plant tissues at cellular or subcellular levels and in response to internal and environmental stimuli during plant development.

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“…Measurements of IAA using an exogenous radioactive target auxin, the exogenous expression of an auxin‐responsive reporter gene, and an immune affinity extraction have been widely used. Alternatively, mass spectrometry (MS)‐based methods provide a specific mass‐to‐charge ratio ( m / z ) that is tightly linked to the chemical structure, and thus enable the direct quantification of IAA.…”

Section: Introductionmentioning

confidence: 99%

Practical optimization of liquid chromatography/mass spectrometry conditions and pretreatment methods toward the sensitive quantification of auxin in plants

Sugahara

Kitao

Yamagaki

et al. 2020

Rapid Comm Mass Spectrometry

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Rationale The plant hormone auxin, indole‐3‐acetic acid, regulates many aspects of plant growth and development. Auxin quantification should offer broad insights into its mechanistic action in plants. However, limited auxin content in plant tissues hampers the establishment of quantification methods without the highest graded instruments or deeply specialized experimental techniques. Methods In this study, we detailed optimized conditions for high‐performance liquid chromatography coupled with triple‐quadrupole mass spectrometry (LC/MS). We compared LC/MS conditions, such as columns, mobile phases, parameters of acquisition methods (selective or multiple ion monitoring), dwell times (DTs), and channel numbers, using differentially mixed authentic auxin and its related compounds. We further investigated pretreatment methods through the optimization of auxin recovery and irrelative compound removal from plant tissues prior to the LC/MS analysis. Results Our LC/MS analysis demonstrated the particular importance of the column, DTs, and channel numbers on detection sensitivity. Our comparative analysis developed optimal pretreatment methods, including the pulverization of plants, concentration of extract through centrifugal evaporation, and removal of irrelative metabolites using liquid–liquid extraction and a spin filter. We injected plant samples into our LC/MS system, quantified auxin and eight related compounds in a single measurement, and determined the auxin increase in an auxin over‐producing mutant. Conclusions Our practical optimization of LC/MS conditions and pretreatment methods provides detailed experimental processes toward the sensitive quantification of auxin from 10 mg of plant tissue. These processes have not always been clearly documented; therefore, our protocol could broadly contribute to technical advances in plant growth and development research.

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What Has Been Seen Cannot Be Unseen—Detecting Auxin In Vivo

Cited by 29 publications

References 216 publications

Auxins and Cytokinins—The Role of Subcellular Organization on Homeostasis

Auxins and Cytokinins—The Role of Subcellular Organization on Homeostasis

New fluorescent auxin probes visualise tissue‐specific and subcellular distributions of auxin in Arabidopsis

Practical optimization of liquid chromatography/mass spectrometry conditions and pretreatment methods toward the sensitive quantification of auxin in plants

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