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.
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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.
Procházkova D, Wilhelmová N (2011) Nitric oxide, reactive nitrogen species and associated enzymes during plant senescence. Nitic oxide 24:61–65CrossRefGoogle Scholar
Gayatri G, Agurla S, Raghavendra AS (2013) Nitric oxide in guard cells as an important secondary messenger during stomatal closure. Front Plant Sci 4:1–11CrossRefGoogle Scholar
Delledonne M, Xia Y, Dixon RA et al (1998) Nitric oxide functions as a signal in plant disease resistance. Nature 394:585–588CrossRefPubMedGoogle Scholar
Agurla S, Gayatri G, Raghavendra AS (2014) Nitric oxide as a secondary messenger during stomatal closure as a part of plant immunity response against pathogens. Nitric Oxide 43:89–96CrossRefPubMedGoogle Scholar
Kojima H, Nakatsubo N, Kikuchi K et al (1998) Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal Chem 70:2446–2453CrossRefPubMedGoogle Scholar
Kojima H, Sakurai K, Kikuchi K et al (1998) Development of a fluorescent indicator for nitric oxide based on the fluorescein chromophore. Chem Pharm Bull (Tokyo) 46:373–375CrossRefGoogle Scholar
Planchet E, Kaiser WM (2006) Nitric oxide (NO) detection by DAF fluorescence and chemiluminescence: a comparison using abiotic and biotic NO sources. J Exp Bot 57:3043–3055CrossRefPubMedGoogle Scholar
Mur LAJ, Mandon J, Cristescu SM et al (2011) Methods of nitric oxide detection in plants: a commentary. Plant Sci 181:509–519CrossRefPubMedGoogle Scholar
Desikan R, Cheung MK, Bright J et al (2004) ABA, hydrogen peroxide and nitric oxide signalling in stomatal guard cells. J Exp Bot 55:205–212CrossRefPubMedGoogle Scholar
Neill S, Barros R, Bright J et al (2008) Nitric oxide, stomatal closure, and abiotic stress. J Exp Bot 59:165–176CrossRefPubMedGoogle Scholar
García-Mata C, Lamattina L (2013) Gasotransmitters are emerging as new guard cell signaling molecules and regulators of leaf gas exchange. Plant Sci 201–202:66–73CrossRefPubMedGoogle Scholar
Kolla VA, Raghavendra AS (2007) Nitric oxide is a signaling intermediate during bicarbonate-induced stomatal closure in Pisum sativum. Physiol Plant 130:91–98CrossRefGoogle Scholar
Srivastava N, Gonugunta VK, Puli MR et al (2009) Nitric oxide production occurs downstream of reactive oxygen species in guard cells during stomatal closure induced by chitosan in abaxial epidermis of Pisum sativum. Planta 229:757–765CrossRefPubMedGoogle Scholar
Gonugunta VK, Srivastava N, Puli MR et al (2008) Nitric oxide production occurs after cytosolic alkalization during stomatal closure induced by abscisic acid. Plant Cell Environ 31:1717–1724CrossRefPubMedGoogle Scholar