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

Abstract

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.

HSP90 affects root growth in Arabidopsis by regulating the polar distribution of PIN1

“…Recently, FluorA I, a 2,4‐D derivative labeled with the 7‐nitro‐2,1,3‐benzoxadiazole fluorophore was developed and used to monitor tissue‐specific and subcellular distributions of auxin (Pařízková et al ., 2021). To test the impact of impaired HSP90 on auxin distribution and transport we applied FluorA I in wild‐type and hsp90 RNAi seedlings.…”

“…These findings suggest that FluorA I is transported in control wild‐type plants, while in roots with impaired HSP90 FluorA I transport is impeded revealing an aberrant distribution and accumulation of auxin in the root. Collectively, our results show a prominent role of HSP90 in the regulation of auxin transport through the control of efflux transporters, as the distribution of FluorA I depends on active auxin efflux transporters (Pařízková et al ., 2021).…”

“…A fluorescent auxin probe FluorA I (Pařízková et al ., 2021), a 2,4‐D derivative labeled with the 7‐nitro‐2,1,3‐benzoxadiazole (NBD) fluorophore, was used to test auxin transport and distribution. 5‐d‐old seedlings were transferred to liquid MS supplemented with 0.4 μΜ FluorA I, then placed on a glass slide and imaged 30 min after FluorA I application using a Zeiss LSM 800 confocal microscope.…”

Endocytic Trafficking Promotes Vacuolar Enlargements for Fast Cell Expansion Rates in Plants

“…To determine the conversion of VAC1 into free PAA, 6-day-old seedlings were treated with DMSO or 10 µM VAC1 for 2.5 h in liquid medium and then flash-frozen in liquid nitrogen. The extraction and quantification methods for both analytes were optimized based on the previously described method (Pařízková et al, 2021). Briefly, the plant samples (∼30 mg FW) were extracted into 100% methanol and purified using liquid-liquid extraction method into 900 µL of extraction solution (methanol:H2O: hexane – 1:1:1), evaporated and dissolved in 50 µL of 100% methanol.…”

“…(B) 6-day-old seedlings were treated with DMSO or 10 µM VAC1 for 2.5 h in liquid medium, flash-frozen in liquid nitrogen and subsequently extracted by liquid-liquid extraction (Pařízková et al, 2021). Quantification of VAC1 and PAA levels was performed by UHPLC-UV-MS system operating in selected ion recording (SIR) mode and calculated from an external calibration using a recovery factor.…”