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Nitric Oxide (NO) Measurements in Stomatal Guard Cells

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1424)

Abstract

The quantitative measurement of nitric oxide (NO) in plant cells acquired great importance, in view of the multifaceted function and involvement of NO as a signal in various plant processes. Monitoring of NO in guard cells is quite simple because of the large size of guard cells and ease of observing the detached epidermis under microscope. Stomatal guard cells therefore provide an excellent model system to study the components of signal transduction. The levels and functions of NO in relation to stomatal closure can be monitored, with the help of an inverted fluorescence or confocal microscope. We can measure the NO in guard cells by using flouroprobes like 4,5-diamino fluorescein diacetate (DAF-2DA). This fluorescent dye, DAF-2DA, is cell permeable and after entry into the cell, the diacetate group is removed by the cellular esterases. The resulting DAF-2 form is membrane impermeable and reacts with NO to generate the highly fluorescent triazole (DAF-2T), with excitation and emission wavelengths of 488 and 530 nm, respectively. If time-course measurements are needed, the epidermis can be adhered to a cover-glass or glass slide and left in a small petri dishes. Fluorescence can then be monitored at required time intervals; with a precaution that excitation is done minimally, only when a fluorescent image is acquired. The present method description is for the epidermis of Arabidopsis thaliana and Pisum sativum and should work with most of the other dicotyledonous plants.

Key words

4,5-Diamino fluorescein diacetate Nitric oxide Reactive nitrogen species Stomatal guard cells Time-course measurement 

Notes

Acknowledgments

Our work on guard cells is supported by grants to ASR of a J C Bose National Fellowship (No. SR/S2/JCB-06/2006) from the Department of Science and Technology and another from Department of Biotechnology, both in New Delhi. We also thank DBT-CREBB, DST-FIST, and UGC-SAP-CAS for support of infrastructure in Department/School.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Plant Sciences, School of Life SciencesUniversity of HyderabadHyderabadIndia

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