A Novel Protocol for Detection of Nitric Oxide in Plants.
Detection of nitric oxide (NO) in plant cells is mostly undertaken using diaminofluorescein (DAF) dyes. Serious drawbacks and limitations have been identified in methods using DAF as a probe for NO detection. The present work reporting an alternative fluorescent probe for NO detection is thus proposed for varied applications in plant systems for physiological investigations. This method involves a simple, two-step synthesis, characterization, and application of MNIP-Cu {Copper derivative of [4-methoxy-2-(1H-napthol[2,3-d]imidazol-2-yl)phenol]} for specific and rapid binding with NO, leading to its detection in plant cells by epifluorescence microscopy and confocal laser scanning microscopy (CLSM). Using sunflower (Helianthus annuus L.) whole seedlings, hypocotyl segments, stigmas from capitulum, protoplasts, and isolated oil bodies, present investigations demonstrate the versatile nature of MNIP-Cu in applications for NO localization studies. MNIP-Cu can detect NO in vivo without any time lag (ex. 330-385 nm; em. 420-500 nm). It exhibits fluorescence both under anoxic and oxygen-rich conditions. This probe is specific to NO, which enhances its fluorescence due to MNIP-Cu complexing with NO and treatment with PTIO leads to quenching of fluorescence. It is relatively nontoxic when used at a concentration of up to 50 μM.
Department of Botany, University of Delhi, North Campus, Delhi, 110007, India.
Prachi
P
Jain
Department of Botany, University of Delhi, North Campus, Delhi, 110007, India.
Anisha
A
David
Department of Botany, University of Delhi, North Campus, Delhi, 110007, India. bhatlasc@gmail.com.
Satish C
SC
Bhatla
D006368:Helianthus / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009569:Nitric Oxide / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D009569:Nitric Oxide
Confocal laser scanning microscopy (CLSM); Copper derivative of 4-methoxy-2-(1H-napthol[2,3-d]imidazol-2-yl)phenol (MNIP-Cu); Diaminofluorescein (DAF); Epifluorescence microscopy; Hypocotyl; In vivo localization; Nitric oxide (NO); Oil bodies; Protoplasts; Stigma papillae; Sunflower; Whole seedlings