Breakthrough Technologies Laser Photoacoustic Detection Allows in Planta Detection of Nitric Oxide in Tobacco following Challenge with Avirulent and Virulent Pseudomonas syringae Pathovars

We demonstrate the use of laser photoacoustic detection (LPAD) as a highly sensitive method to detect in planta nitric oxide (NO) production from tobacco (Nicotiana tabacum). LPAD calibration against NO gas demonstrated a linear relationship over 2 orders of magnitude with a detection threshold of ,20 pmol h (1 part per billion volume [ppbv]). The specificity of the photoacoustic signal for NO when adding gas or the NO donor, sodium nitroprusside, on injection into plant leaves, was demonstrated by its abolition with O3 ( NO 1 O3 / NO2 1 O2). The utility of the LPAD method was shown by examination of a nonhost hypersensitive response and a disease induced by Pseudomonas syringae (P. s.) pv phaseolicola and P. s. pv tabaci in tobacco. NO was detected within 40 min of challenge with P. s. pv phaseolicola, some 5 h before the initiation of visible tissue collapse. The wildfire tobacco pathogen P. s. pv tabaci initiated NO generation at 2 h postinfection. The use of NO donors, the scavenger CPTIO ([4-carboxyphenyl]-4,5-dihydro-4,4,5,5-tetramethyl-3-oxide), and the mammalian nitric oxide synthase inhibitor L-NMMA (N-monomethyl-L-arginine) indicated that NO influenced the kinetics of cell death and resistance to both avirulent and virulent bacteria in tobacco. These observations suggest that NO is integral to the elicitation of cell death associated with these two bacterial pathogens in tobacco.