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Visualizing plant salt stress with a NaCl-responsive fluorescent probe

Nature Protocols volume 20, pages 902–933 (2025)Cite this article

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An Addendum to this article was published on 06 October 2025

Abstract

Salt stress is an adverse environmental condition that harms plant growth and development. The development of salt stress probes is critical for tracking the growth dynamics of plants, molecular breeding or screening of growth regulators. The sodium chloride (NaCl)-responsive fluorescent probe Aza-CyBz is designed based on the tenet that NaCl induces formation of ordered aggregates, and the sensitive fluorescence response can enable the visualization of plant salt stress in root tip tissues and live plants. Herein, we describe a detailed three-step route for synthesis of Aza-CyBz and applications to monitoring salt stress in Arabidopsis thaliana. The procedures for operating fluorescence imaging under various stresses are also listed to eliminate interference from the oxidative mechanism of salt stress. Compared with conventional invasive approaches such as inductively coupled plasma emission spectrometry and flame photometer, our protocol can real-time monitor salt stress experienced by plants, which demands simple pretreatment procedure and staining technique. Due to near infrared fluorescence, this method provides direct visual observation of salt stress at both tissue and live plant levels, which is superior to conventional noninvasive approaches. The preparation of probe Aza-CyBz takes ~2 d, and the imaging experiments for assessing salt stress experienced by plants, including the preparation of stressed plant samples takes ~9–11 d for root tip tissues and ~23 d for live plants. Notably, acquisition and analysis visual images of salt stress in plants can be completed within 2 h and they require only a basic knowledge of spectroscopy and chemistry.

Key points

  • It is useful to measure NaCl uptake in plants noninvasively, e.g., to discover variants with better salt tolerance. This protocol describes how to prepare and use a NaCl-responsive fluorescent probe Aza-CyBz to image Arabidopsis root tips or whole plants.

  • The probe itself has an interesting mechanism: the presence of NaCl results in its aggregation and a reduction in fluorescence. The near infrared fluorescence could avoid background fluorescence interference from chlorophyll.

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Fig. 1: Visualization of salt stress in root tips.
Fig. 2: Procedures for the visualization of salt stress of whole plants in vivo and control experiments in a Petri dish, with the time arrows indicating the duration of each step.
Fig. 3: Synthesis route and application of NaCl-responsive heptamethine cyanine Aza-CyBz.
Fig. 4: Spectroscopic characterization of Aza-CyBz toward NaCl.
Fig. 5: Fluorescence imaging of Aza-CyBz for plant salt stress in root tips.
Fig. 6: Fluorescence imaging of Aza-CyBz for the visualization of salt stress in live plants.
Fig. 7: Control experiments for the validation of NaCl sensing in plants with Aza-CyBz.
Fig. 8: Control imaging experiments with commercial CoroNa Green probe in vivo.

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Data availability

All supporting data are included in the article, its supplementary information, the supporting research paper and figshare. When the plants were photographed, each photograph had three plants. The images were cropped to show individual plants. In some cases, the cropped photographs still contained parts of other plants and these were hidden using black background. All unedited versions of main and supplementary figures can be found in figshare (https://doi.org/10.6084/m9.figshare.28985144).

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Acknowledgements

This work was supported by the National Key R&D Program of China (2022YFA1207400), National Natural Science Foundation of China (22274061), the 111 Project (B17019) and Fundamental Research Funds for the Central Universities (CCNU24JCPT030). We would like to thank Y. Guo and G. Liu from China Agricultural University and H. Wu and J. Li from Huazhong Agricultural University for their guidance and assistance.

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Authors and Affiliations

  1. State Key Laboratory of Green Pesticide, International Joint Research Center for Intelligent Biosensor Technology and Health, College of chemistry, Central China Normal University, Wuhan, P. R. China

    Xiaoxie Ma, Xiaoyan Zeng, Yurou Huang, Sheng Hua Liu, Jun Yin & Guang-Fu Yang

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  1. Xiaoxie Ma
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  2. Xiaoyan Zeng
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  3. Yurou Huang
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  5. Jun Yin
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Contributions

All the experiments were conducted and the manuscript was written by X.M. with the supervision of S.H.L., J.Y. and G.-F.Y. X.Z. and Y.H. contributed to the manuscript writing.

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Correspondence to Jun Yin or Guang-Fu Yang.

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Nature Protocols thanks Sergey Shabala and the other, anonymous, reviewer(s) for their contribution to the peer review process of this work.

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Ma, X. et al. Angew. Chem. Int. Ed. 62, e202216109 (2023): https://doi.org/10.1002/anie.202216109

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Ma, X., Zeng, X., Huang, Y. et al. Visualizing plant salt stress with a NaCl-responsive fluorescent probe. Nat Protoc 20, 902–933 (2025). https://doi.org/10.1038/s41596-024-01068-x

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