Fluorescence‐activated multi‐organelle mapping of subcellular plant hormone distribution

• Published in: The Plant journal : for cell and molecular biology Vol. 116; no. 6; pp. 1825 - 1841
• Main Authors: Skalický, Vladimír, Antoniadi, Ioanna, Pěnčík, Aleš, Chamrád, Ivo, Lenobel, René, Kubeš, Martin F., Zatloukal, Marek, Žukauskaitė, Asta, Strnad, Miroslav, Ljung, Karin, Novák, Ondřej
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.12.2023
• Subjects: Allocations; Arabidopsis - metabolism; Arabidopsis thaliana; Auxin; Auxins; Botanik; Botany; Cell Biology; Cellbiologi; cytokinin; Cytokinins; Cytokinins - metabolism; Developmental plasticity; Endoplasmic reticulum; Endoplasmic Reticulum - metabolism; Flow cytometry; Fluorescence; Fractionation; Gene Expression Regulation, Plant; Homeostasis; Hormones; Hormones - metabolism; Indoleacetic Acids - metabolism; LC–MS/MS; Light scattering; Localization; Organelles; Phytohormones; Plant growth; Plant Growth Regulators - metabolism; Plant Roots - metabolism; subcellular fractionation; subcellular homeostasis; technical advances; Vacuoles; Arabidopsis thaliana; LC-MS/MS; subcellular homeostasis; Auxin; cytokinin; subcellular fractionation; technical advances; flow cytometry
• ISSN: 0960-7412; 1365-313X; 1365-313X
• DOI: 10.1111/tpj.16456

SUMMARY Auxins and cytokinins are two major families of phytohormones that control most aspects of plant growth, development and plasticity. Their distribution in plants has been described, but the importance of cell‐ and subcellular‐type specific phytohormone homeostasis remains undefined. Herein, we revealed auxin and cytokinin distribution maps showing their different organelle‐specific allocations within the Arabidopsis plant cell. To do so, we have developed Fluorescence‐Activated multi‐Organelle Sorting (FAmOS), an innovative subcellular fractionation technique based on flow cytometric principles. FAmOS allows the simultaneous sorting of four differently labelled organelles based on their individual light scatter and fluorescence parameters while ensuring hormone metabolic stability. Our data showed different subcellular distribution of auxin and cytokinins, revealing the formation of phytohormone gradients that have been suggested by the subcellular localization of auxin and cytokinin transporters, receptors and metabolic enzymes. Both hormones showed enrichment in vacuoles, while cytokinins were also accumulated in the endoplasmic reticulum. Significance Statement We introduce a breakthrough technique, so‐called Fluorescence‐Activated multi‐Organelle Sorting (FAmOS), for simultaneous fractionation of up to four organelle populations (nuclei, chloroplasts, mitochondria and endoplasmic reticulum) from a single sample of plant cell cultures. The unique combination of our developed FAmOS tool with ultra‐sensitive mass spectrometry‐based analytical methods enables high‐resolution mapping of plant metabolites in isolated organelles.