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BREAKTHROUGH TECHNOLOGIES

Laser Photoacoustic Detection Allows in Planta Detection of Nitric Oxide in Tobacco following Challenge with Avirulent and Virulent Pseudomonas syringae Pathovars¹

Institute of Biological Sciences, University of Wales, Aberystwyth, SY23 3DA, Wales, United Kingdom (L.A.J.M., N.J.H., A.R.S.); and Life Science Trace Gas Exchange Facility, Department of Molecular and Laser Physics, Catholic University of Nijmegen, 6525 ED Nijmegen, The Netherlands (I.E.S., L.J.J.L., F.J.M.H.)

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 (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 + O₃ NO₂ + 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^G-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.