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Plant Science 181 (2011) 509– 519

Contents lists available at ScienceDirect

Plant Science

jo u rn al hom epa ge: www.elsev ier .com/ locate /p lantsc i

eview

ethods of nitric oxide detection in plants: A commentary

uis A.J. Mura,∗, Julien Mandonb, Simona M. Cristescub, Frans J.M. Harrenb, Elena Pratsc

University of Wales, Aberystwyth, Institute of Biological Sciences, Aberystwyth, Wales, UKLife Science Trace Gas Facility, Radboud University Nijmegen, Heijendaalseweg 135, 6525 AJ Nijmegen, The NetherlandsInstituto de Agricultura Sostenible, CSIC, Alameda del Obispo, Menéndez Pidal s/n, 14080 Córdoba, Spain

r t i c l e i n f o

rticle history:eceived 10 March 2011eceived in revised form 5 April 2011ccepted 6 April 2011vailable online 13 April 2011

eywords:itric oxide measurement

n/Ex planta assaysriess reagent

a b s t r a c t

Over the last decade nitric oxide (NO) has been shown to influence a range of processes inplants. However, when, where and even if NO production occurs is controversial in several phys-iological scenarios in plants. This arises from a series of causes: (a) doubts have arisen over thespecificity of widely used 4,5-diaminofluorescein diacetate (DAF-2DA)/4-amino-5-methylamino-2,7-difluorofluorescein (DAF-FM) dyes for NO, (b) no plant nitric oxide synthase (NOS) has been cloned, sothat the validity of using mammalian NOS inhibitors to demonstrate that NO is being measured is debat-able, (c) the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (cPTIO)needs to be used with caution, and (d) some discrepancies between assays for in planta measurementsand another based on sampling NO from the gas phase have been reported. This review will outline some

iaminofluorescein (DAF) dyesO electrodeslectron spin resonanceass spectrometry

hemiluminescenceaser photoacoustic detection

commonly used methods to determine NO, attempt to reconcile differing results obtained by differentlaboratories and suggest appropriate approaches to unequivocally demonstrate the production of NO.

© 2011 Elsevier Ireland Ltd. All rights reserved.

uantum cascade laser

ontents

1. Nitric oxide in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5102. In planta assays for NO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510

2.1. The oxyhemoglobin assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5102.2. The Griess reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5102.3. Diaminofluorescein (DAF) fluorescent dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5102.4. Electron spin resonance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5122.5. NO electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5132.6. Mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513

3. Ex planta assays for NO: detection of gaseous NO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5133.1. Chemiluminescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513

3.2. Laser based infrared spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.2.1. CO laser-based photoacoustic detection . . . . . . . . . . . . . . . . .

3.2.2. Quantum cascade lasers (QCL) based detection. . . . . . . . . .

Abbreviations: AA, ascorbic acid; BY-2, Bright Yellow-2; cPTI, 2-(4-carbo,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide; DAF, diaminofluorescein; DAF-2DA, 4,-amino-5-methylamino-2,7-difluorofluorescein; DAF-FM-T, 4-amino-5-methylamino-2etic resonance; ESR, electron spin resonance; fwt, fresh weight; Hb, hemoglobin; HbO2, oFC, mass flow controller; MIMS, membrane inlet mass spectrometry; MetHb, methemo

ase; NED, N-(1-naphthyl) ethylenediamine; NOS, nitric oxide synthase, P01, Pallas 01; PMy volume; PTFE, polytetrafluoroethylene; QCL, quantum cascade lasers; RIMS, restriction∗ Corresponding author. Tel.: +44 01970 622981.

E-mail address: lum@aber.ac.uk (L.A.J. Mur).

168-9452/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.plantsci.2011.04.003

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515

xyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl; cPTIO, 2-(4-carboxyphenyl)-5-diaminofluorescein diacetate; DAF-2T, diaminofluorescein triazole; DAF-FM,,7-difluorofluorescein triazole; DHA, dehydroascorbic acid; EPR, electron paramag-xyhemoglobin; HR, hypersensitive response; LAPD, laser photoacoustic detection;globin; NADPH, nicotinamide adenine dinucleotide phosphate; NR, nitrate reduc-T, photomultiplier tube; ppbv, part-per-billion by volume; ppmv, parts per million

capillary inlet mass spectroscopy; ST, spin-trap.

510 L.A.J. Mur et al. / Plant Science 181 (2011) 509– 519

4. NO measurements—some recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518

. . . . . .

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2

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Nitric oxide in plants

Although there have been suggestions of roles for NO in plantsor many decades [1], it was only in the 1990s that a pioneeringeries of articles by Leshem [2], Delledonne et al. [3] and Durnert al. [4] clearly established that this was truly a signal moleculen plants. Merely a cursory glance through this special issue willuickly illustrate how NO has emerged as an important signal inlants. However, the most appropriate method to measure NO pro-uction in plants is still controversial. Definitive NO measurementsre required to actually establish that it is being produced within

given biological context. Moving on from this, the kinetics of NOroduction must be determined to set its generation within theontext of physiological/cytological/genetic events and the pres-nce of other signals. Furthermore, appropriate treatments withither NO gas or NO donors can be used in such as transcriptomicxperiments [5] or proteomic-based identification of S-nitrosylatedr nitrated proteins [6,7].

Problems have arisen for a number of reasons, mostly due thehysical properties of NO itself. In the atmosphere the half-life ofxidation is dependent on the NO concentration [8]. At concentra-ions of 100 part per million volume (ppmv) or greater the half-lifeor oxidation of NO to NO2 by atmospheric oxygen is 1 h or lessespectively, whereas at low concentrations (∼0.01 ppmv) the half-ife for this oxidation is of the order of 10,000 h. Furthermore, theiological effects of NO are concentration dependent [9,10], whichemand that NO must be measured over a broad range of concen-rations (pM to mM) to determine its action. Rates of NO productionary enormously with measurements of 0.1–∼200 nmol/h/g fwteing reported [11–13]. NO production may also be restricted toery few cells, for example in guard cells [14]. Thus, measure-ent methods must be very sensitive to be able to detect NO

roduction from plants. In addition, significant doubts have beenxpressed as to the specificity of the detection methods such ashe use of DAF dyes [15] which are employed by large numbersf NO researchers. This review will briefly describe some of theany available methods to detect NO and consider their advan-

ages and disadvantages. In doing so, we will not attempt to providen encyclopaedic description of the many methods through whichO may be measured but concentrate on those which have beensed by plant scientists. We also will not be considering nitric oxideynthase assays through which estimations of NO production canade by measuring the oxidation of arginine to form citrulline. This

ndirect assay has contributed to making NO measurements quiteoutine in animal systems. However, in the absence of a plant NOSene, we do not wish to consider the various “NOS-like” mecha-isms through which plants could generate NO and may, or mayot, be confounding the use of this assay in plants. Finally, we willuggest some common approaches that could be followed to yieldobust measurement of NO production.

. In planta assays for NO

Many assays focus on determining NO content within plant tis-ues to assess the actual concentration that impacts on cellularrocesses and physiology.

.1. The oxyhemoglobin assay

Early papers on plant NO production utilised a hemoglobinased assay to measure NO production [3,16]. This is a spectro-

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518

scopic method based on the reaction of oxyhemoglobin (HbO2)with NO to produce methemoglobin (MetHb) and nitrate (NO3)[17]. This reaction results in a shift of absorbance from 415–421 nm(HbO2) to 401 nm (MetHb). This is a robust and sensitive assay witha predicted detection limit of 1.3–2.8 nM [18].

However, recently this technique has fallen out of favour for aseries of reasons. Firstly, the production of fresh HbO2 is techni-cally demanding, as it requires hemoglobin oxygenation followedby isolation using chromatography. More seriously, reactive oxygenspecies can also oxidise HbO2 to give false readings from the assay.Delledonne et al. [3] applied catalase and superoxide dismutase totheir assays to suppress ROS production but, although possessingan extraordinary high catalytic activity (kcat s−1 40,000,000), thelow affinity (Km 25 mM) of catalase for its substrate means thatthe presence of H2O2 could remain a confounding factor. This isparticularly problematic since the production of NO occurs simul-taneously, or near simultaneously, with that of H2O2 during plantdefence responses. Additionally, changes in pH – also a feature ofplant defence responses – can affect the assay as can the presenceof competing heme containing proteins.

2.2. The Griess reaction

The Griess reaction is one of the most widely used assays for NOdetection and represents the basic reaction of relatively cheap com-mercial kits for NO measurements. The technique was pioneered byJohann Peter Griess (1829–1888), a German organic chemist, whowas one of the founders of the azo and diazo dye industry. Griesssuggested that nitrites could be detected by reacting with sul-phanilic acid and �-naphthylamine under acidic conditions to yieldan azo dye. This remains the basic reaction except that today sul-phaniliamide and N-(1-naphthyl) ethylenediamine (NED) are usedto react with NO2. The resulting stable water-soluble azodye may bequantified by measuring spectrometric absorption at 520 nm. NOcan be readily oxidized to NO2 (usually by CrO3) so that the basicGriess reaction is used as an indirect assay for NO (Fig. 1). NO2 canbe further oxidized to NO3 which does not form the azodye butthe kinetics of NO2 oxidation are relatively slow and are thereforeconsidered to be insignificant [19].

The popularity of the Griess reaction for determining NO inclinical and animal research [20–23] has not been mirrored plantscience. The Griess assay has been used to determine nitrite ionsin cucumber, tomato and wine [24,25] and perhaps most impor-tantly by Vitecek et al. to measure NO production from tobacco(Nicotiana tabacum) cultures inoculated with the cell death elicitor,cryptogein, and Arabidopsis mutants exhibiting either increasedor decreased NO synthesis [26]. The Vitecek et al., study clearlydemonstrated the potential of the Griess reagent, so that therelative lack of interest from plant scientists is worthy of brief com-ment. It may be that its reported lack of sensitivity at 0.5 �M NO[27,28] may be deterring its use. However, through a novel imple-mentation of the Griess reaction developed by Vitecek et al. [26]sensitivities in the nM range were reported. It seems much morelikely therefore that the attractiveness of the use of DAF dyes requir-ing only the use of fluorescent (ideally confocal) microscopes hasdistracted plant researchers.

2.3. Diaminofluorescein (DAF) fluorescent dyes

DAF dyes have been very widely used by plant NO scientists(including ourselves [Fig. 2]) to reveal likely sites of NO genera-

L.A.J. Mur et al. / Plant Science 181 (2011) 509– 519 511

Fig. 1. Griess reagent method of NO detection. (A) Nitric oxide (NO) is oxidised to nitrite (NO2−), by CrO3. NO2

− reacts with sulphanilamide to form a diazonium saltintermediate. The diazonium salt is then coupled to N-(1-naphthyl) ethylenediamine (NED) to form the stable water-soluble azo dye (�max 540 nm). (B) Schematic of thea velopc ns sulpt

tscNeo

tdwabfrflt[bc

wttdcotw

pparatus used to detect NO in the gaseous phase based on the Griess reaction as dehamber. Any NO2 (or HNO2) in the airflow is captured in the first trap which contaihe NO to NO2 which is detected by a second Griess reaction ion trap 2.

ion [29–33]. DAF dyes can be readily obtained from commercialources at a reasonable price and NO can be visualised via fluores-ence microscopy. Superficially, it also appears easy to prove thatO is being generated; simply co-apply DAF with either NO scav-ngers (for example, cPTIO; NO + cPTIO → NO2 + cPTI) or inhibitorsf mammalian NOS.

DAF dyes were first described by Kojima et al. [34,35] wherehey were shown to react with N2O3, a by-product of NO oxi-ation, with a resulting dramatic increase in fluorescence. Thisas initially commercialised in a diacetate-form (DAF-2DA) which

llowed ready uptake by living cells. The diacetate group is removedy cellular esterases leaving the membrane impermeable DAF-2orm available for nitration by N2O3 to generate the highly fluo-escent triazole (DAF-2T; Fig. 2A) [39]. Encouragingly, no DAF-2Tuorescence was observed with NO2

−, NO3−, H2O2 and peroxyni-

rite (ONOO−), and very low detection limits at 5 nM were reported35]. This dye may be used in flow cytometry [36] but have mostlyeen used to image patterns of cellular NO production by fluores-ence microscopy.

Very soon after their development the specificity of DAF dyesas challenged from various quarters. It may be predicted that

he antioxidant ascorbic acid should reduce levels of N2O3 andherefore the DAF-2T signal, but actually DAF2 reacts with dehy-roascorbic acid (DHA) and ascorbic acid (AA) to generate new

ompounds that have fluorescence emission profiles similar to thatf DAF-2T [37]. This problem can only be partially solved throughhe use of ascorbate oxidase where AA is reduced to DHA andater [38], which is also impractical when attempting to mea-

ed by Vitecek et al. [26]. A gas flow passes through a humidifier and into the samplehanilamide and NED but NO progresses into the oxidiser tube where CrO3 oxidizes

sure in planta NO where cellular penetration of the enzyme canbe expected to be negligible. However, it may be that concentra-tions of >5 mM AA are required to elicit a detectable fluorescencesignal [39]. Planchet and Kaiser [39] have also noted fluorescenceunder anoxic conditions, which should not be possible given thedependence on the oxidation of NO to N2O3; and although, thiscould not be suppressed with cPTIO it appeared to be dependenton nitrate reductase activity. Other problems include the differen-tial loading of DAF dyes into different tissues and association withnon-NO producing dead cells [26].

DAF-FM has been developed as more sensitive NO sensor toDAF-2DA (respectively, ∼3 nM and ∼5 nM [40]). It has also beensuggested that the fluorescent signal of DAF-FM is not affected bypH above 5. However, when measuring the concentration of dis-solved NO in water using DAF-FM, Vitecek et al. [26] noted that thefluorescence obtained with 300 nM NO was quenched with increas-ing pH so that the signals at pH 9 were around half those obtainedat pH 5.5. The plant cell cytosol pH is usually around 7.5, with theapoplast and vacuole being in the region of pH 5.5, but intracellularpH can change dramatically during cellular processes such as thepathogen-elicited HR, root tip growth, nodulation, gibberellic acidand abscisic acid signalling [41–43]. Thus, potential pH changesshould be considered when intepreting DAF-FM result.

Many of the researchers that used DAF dyes confirmed NO detec-

tion with cPTIO that scavenge NO and consequently suppress DAFfluorescence. However, this three-way interaction between NO,cPTIO and DAF-2 is not straight forward as its outcome dependson the relative concentration of all three reactants. cPTIO is a

512 L.A.J. Mur et al. / Plant Science 181 (2011) 509– 519

Fig. 2. Use of DAF dyes to suggest localised generation of NO in powdery mildew (Blumeria graminis f.sp. hordei) challenged Barley (Hordeum vulgare cv Pallas 01 [P-O1])and Arabidopsis thaliana Col-0. (A) On application 4,5-diaminofluorescein diacetate (DAF-2DA) is readily taken up into cells where cytoplasmic esterases remove the acetategroups (to generate 4,5-diaminofluorescein; DAF-2) preventing movement back out of the cell. DAF-2 can react with N2O3, an oxidation product of NO to generate the highlyfluorescent DAF-2T (triazolofluorescein). (B) Background auto fluorescence and (C) fluorescence on treatment with DAF-2DA in barley (Hordeum vulgare cv Pallas 01 [P-01]harbouring resistance gene Mla1) at sites of attack with powdery mildew (Blumeria graminis f. sp. hordei race CC1) at 15 h following infection. Arrowed is a cell undergoinga hypersensitive response (HR). All other sites of fluorescence are associated with developing papillae and a stoma. Bar = 50 �m. (D) Site of attack in Arabidopsis thalianaby B. graminis f. sp. hordei race CC1 at 15 h following infection. Following application of DAF-2DA fluorescence at the site of papilla formation is arrowed (red). The papillai s out ot = 50 �e

s[(NfiocsecHtbeab

2

ain[

s occurring on the underside of an appressorial germ tube. The condium (spore) iargeting. Fluorescence is also observed in the stomatal guard cells (blue arrow). Bart al. [30].

table organic radical that was developed by Akaike and Maeda44] and oxidizes the NO molecule to form the

•NO2 radical

NO + cPTIO → •NO2 + CPTI).

•NO2 radical can react with NO to form

2O3 (NO2 + NO → N2O3) which in turn can react with DAF-2 toorm fluorescent DAF-2T. This could suggest that cPTIO shouldncrease fluorescence; however, this ignores the competing directxidation of NO (4NO + O2 → 2N2O3) to form N2O3. Thus, at higherPTIO concentrations NO will be rapidly converted to

•NO2 thereby

lowing the formation of N2O3. This third order dependence likelyxplains the failure of Vitecek et al. [26] to suppress the fluores-ence of DAF-FM in the presence of 380 nM NO with 100 �M CPTIO.owever, worryingly, when DAF-FM was allowed to react with NO

o form DAF-FM-T (i.e. relatively little free NO), cPTIO proved toe effective masking fluorescence; a feature also observed by Aritat al. [45]. Until the exact nature of this reaction is understood, ide-lly, cPTIO should not be the only control used by researchers (seeelow).

.4. Electron spin resonance

Electron spin resonance ([ESR], also known as electron param-

gnetic resonance [EPR]) is based on observing unpaired electronsn magnetic fields which in the microwave region exhibit a “reso-ance” between parallel and antiparallel electron spin orientations46]. ESR instruments will scan the magnetic field strength until

f focus but indicated by a dotted oval. Note that the papilla is the focus of vesiclem. DAF-2DA treatment methods and confocal microscopy were as detailed in Prats

resonance between the parallel and antiparallel states is reachedat a given microwave frequency (which will be specific to a givenradical) until a signal is observed. As EPR only detects free radi-cal species, it is highly selective to NO over all other products ofN oxidation [46]. However, the ephemeral nature of the NO rad-ical entails using specific “spin-trap (ST)” chemicals which givelongevity to a NO-dependent radical signal (NO. + ST → NO–ST).The detection limits of ESR are in the order of pmol [47]. Iron-dithiocarbamates have been often used for ESR which exploits thehigh affinity of NO for iron [48]. Iron-dithiocarbamates ST (Fe(S2CN-R R′)2), exist with a range of side groups (R and R′ can be eitherbe methyl-, ethyl-, glucamine-, sarcosine- or amino acids) [47].The different properties conferred by these side groups are usefulfor targeting to for example, hydrophobic membranes in the caseof Fe-diethyldithiocarbamate or extracellular fluids with the polarFe-N-methyl-d-glucamine dithiocarbamate [46].

ESR has been used in plants to report NO production frompollen [49], sorghum embryonic axes [50] and also Arabidopsisinfected with bacterial pathogens [51]. Further, lipophillic ST hasbeen used to show NO effects on the oxygen-evolving complexof photosystem II from cyanobacterium Synechococcus elongates

[52]. In a particularly interesting paper, Cao et al. [53] demon-strated the detection of NO and reactive oxygen species followingthe co-application of different ST. Such successes notwithstand-ing, EPR has not been widely used by plant scientists due to

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L.A.J. Mur et al. / Plant

he inherent costs of EPR resonators and the considerable exper-ise required to exploit this platform; such that most biologicaltudies involve collaborations with physics departments. Experi-entally, EPR whilst excellent for one-off readings is difficult to

pply to continuous, long term, reading of the same plant sample54].

.5. NO electrodes

NO electrodes have been widely used by clinical scientists ashey represent a relatively cheap and easy means to detect NO55]. The “classical” NO electrode consists of a platinum/Teflonoated working electrode and a Ag/AgCl reference electrode, bothncased in a glass micropipette filled with 30 mM NaCl/0.3 nM HClolution except for an open end covered with and NO-permeableembrane. These can be made from different compounds such

s chloroprene rubber, cellulose acetate, collodion/polystyrene,olytetrafluoroethylene (PTFE), and phenylenediamine [55]. Uponassage of an electric current NO is detected based on its oxida-ion at +0.8 to +0.9 V compared to the reference electrode [56].eported sensitivities of NO electrodes have been in the orderf 10−20 mol of NO in single cells [57]. The cost of commerciallyvailable NO sensors could be prohibitive to some groups butesson-Bard et al. [58] constructed an inexpensive homemade plat-

num/iridium (Pt/Ir) microelectrode to measure NO. The Pt/Ir wireas insulated with Teflon PTFE and the wire cut at end to form

disc on to which first nickel phthalocyanine and then Nafiona fluoropolymer-copolymer) and finally o-phenlenediamine wasrecipitated (for details see [58]). This proved to a sensitive andpecific probe for measuring NO production in cryptogein treatedobacco cell cultures.

Leshem [2] demonstrated that an NO electrode could be used inlants to detect NO simply pushing the electrode into fruit. How-ver, several plant organs are not amendable to such intervention;hus, electrodes have been most often used in plant tissue culture.lectrodes have been used to reveal NO production during cad-ium induced cell death in tobacco BY-2 cells [77] and cultures of

reen alga Chlamydomonas reinhardtii [59]. In cryptogein treatedobacco plants Besson-Bard et al. [58] shown that the signals fromn NO electrode were not affected by physiologically relevant levelsf nitrate, O2

−, ONOO− or H2O2. In a very interesting in planta studyO microelectrodes were inserted into pelargonium leaves and thisllowed the detection of the rapid generation of NO within minutes

f wounding followed a second wave at 2 h. Judicious positioning ofhe microelectrodes revealed that NO generation was restricted tohe site of injury [60]. Clearly, this study shows the possible widertility of NO electrodes in plant science.

ig. 3. Chemiluminescent detection of nitric oxide. Schematic diagram of a chemilumineschere NO production is occurring and then on to the reaction cell within the NO analyzer

ell, NO reacts with ozone (O3) to form excited-state nitrogen dioxide (NO2*), which emitsn optical filter and is amplified in a photomultiplier tube (PMT) and quantified.

e 181 (2011) 509– 519 513

2.6. Mass spectrometry

Another method surprisingly neglected by plant scientistswas described by Conrath et al. [69]. This mass spectromet-ric approach allowed the on-line detection of NO from eithertissue cultures or whole plants. In membrane inlet mass spec-trometry (MIMS), a membrane separates the mass spectrometerfrom the tissue culture, but allows the diffusion of small molec-ular weight gases, such as NO. MIMS was used to detect NOproduction from tissue cultures of either tobacco or soybean inoc-ulated with HR-eliciting or disease forming strains of Pseudomonassyringae. In a restriction capillary inlet MS (RIMS) configuration,NO was sampled in the gaseous phase from cuvettes sprayedwith 20 mM NaNO3. A particular attractiveness of RIMS/MIMSis that it is able to distinguish between different N isotopes, sothat on supplementation of (for example) cultures with likelysubstrates for NO generating enzymes (for example N15-labellednitrate/nitrites/polyamines/hydroxylamines) their contribution (ifany) to NO production can be estimated. Given the prevalenceof MS infrastructure in many Institutes and Universities, thereshould be many opportunities for plant NO scientists to exploit theRIMS/MIMS approaches.

3. Ex planta assays for NO: detection of gaseous NO

Although NO is readily soluble in water (7.4 mL/100 mL), it eas-ily volatilises into the gaseous phase (critical temperature: −93 ◦C;critical pressure: 64.85 bar). Thus, a range of approaches haveemerged to measure gaseous NO concentrations, which are attrac-tive as they can provide on-line, in planta measurements of thekinetics of NO production. However, these approaches should, ofnecessity, be seen as only an indicator of in planta NO production,as sampling from the atmosphere represents “lost” NO in terms ofplant signalling. In all of the approaches described below it shouldalso be noted that the possibility of artifactual readings arisingfrom other volatiles – in many cases water vapour – needs to beconsidered.

3.1. Chemiluminescence

By far the most well-established approach to measure gaseousNO is the chemiluminescent assay (Fig. 3), which is based on itsreaction with O3 to yield light photons. This is a two stage reac-

tion whereby the reaction of NO with O3 produces excited-statenitrogen dioxide (NO2*), which emits a photon upon relaxationto the ground state: the emitted light, at >600 nm wavelength,is measured with a photomultiplier tube (PMT) with its inten-

ence-based nitric oxide (NO) assay. A carrier gas is passed through a sample cuvette (the Sievers Nitric Oxide Analyzer [NOA 280i] analyzer is depicted). In the reaction

a photon when relaxing to its ground state (NO2). The emitted light passes through

514 L.A.J. Mur et al. / Plant Science 181 (2011) 509– 519

Fig. 4. NO detection in the gaseous phase using laser photoacoustic detection (LAPD). (A) Plant material [here shown as a red pepper fruit] is carried on an airflow (inthe region of 1–2.5 L/h) by mass flow controllers (not shown). Water vapour in the gas flow is removed using a Peltier cooling element (−5 ◦C) and a cold trap (−80 ◦C)(not shown), prior to passage into the photoacoustic cell. The photoacoustic cell was inserted in a laser cavity, to improve laser power and thus detection sensitivity. Togenerate a photoacoustic signal the laser light was modulated by a chopper (modulation frequency 1000 Hz). In the case of NO, absorption and relaxation in response toc ). S is proportional to the NO gas concentration via a photoacoustic cell constant (F), them hat laser frequency, all of which are constant; as well as laser power (P) which is known.( rred to the web version of the article.)

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Fig. 5. Detection of nitric oxide using a quantum based laser. (A) A quantum cascadelaser (QCL) emitting around 1850 cm−1 passes through a multi-pass cell, NO releasedby the biological samples is transported with a carrier flow of air via gas tubing to

hopped laser light (at infrared wavelength) generates the photoacoustic signal (Sicrophone sensitivity (Sm) and the infrared absorption strength (a) of the gas at t

For interpretation of the references to color in this figure legend, the reader is refe

ity is proportional to the amount of NO (Fig. 3). The results areighly specific for NO. Although chemiluminescence can also result

rom the reaction of O3 with ethylene and sulphur compounds;hese reactions emit at 440–470 and <400 nm, respectively, muchower than the specific NO/O3 reaction. The chemiluminescencepproach exhibits excellent sensitivity with limits of detection asow as 20–50 pmol [61] and need only minimal equipment whichas contributed to its commercialisation as robust platform for NOeasurement. Within plant science the chemiluminescent plat-

orm has been used to measure NO during anoxia [13] synthesis ofO from hydroxylamines [62] and the NADPH-dependent reduc-

ion of nitrite to NO in mitochondria via a non-NR mechanism63].

.2. Laser based infrared spectroscopy

.2.1. CO laser-based photoacoustic detectionNO may also be measured using techniques that are based on

nfrared (IR) absorption. These approaches make use of the specificbsorption of NO at 5.3 �m (∼1876 cm−1) at the infrared wave-ength region [64]. Two platforms will be discussed here; laserhotoacoustic detection (LAPD) and quantum cascade lasers (QCL)ased systems.

LPAD is based on the detection of evolved gases as they adsorbapidly chopped infrared light. The resulting absorption-relaxationesults in pressure variations to generate sound at the frequencyf the modulation by the chopper, which is detected by a micro-hone located within the photoacoustic cell. The photoacoustic cell

s placed inside the cavity of the laser to achieve a high effectiveaser power. The strength of the photoacoustic signal (“sound”)enerated in the cell depends on the property of the gas, i.e. thebsorption coefficient and is proportional to the concentration ofbsorbing trace gas molecules (Fig. 4) [65].

NO detection by LAPD involves the use of a CO laser whose

mission covers the spectral region from 4.6 to 8.2 �m. Fol-owing up pioneering work by [66], our use of LAPD involved

easuring the photoacoustic signal over five laser lines (at933.4, 1921.8, 1900.0, 1856.4, and 1790.6 cm−1) to calculate

the multi-pass cell at a flow rate of 1 L/h. The intensity of the laser (I0) is stronglyattenuated due to the NO absorption of the light in the multi-pass cell (effective pathlength L = 76 m), following the Beer–Lambert law. The detected signal (I) depends ofthe laser intensity (I0), the absorption length (L) and the absorption coefficient. TheNO concentration is calculated by measuring the attenuation of the light (I/I0).

L.A.J. Mur et al. / Plant Science 181 (2011) 509– 519 515

Fig. 6. Comparison of quantum cascade laser (QCL) and chemiluminescent based nitric oxide detection platforms. (A) A 2-week-old tomato (Solanum esculentum) mutantsitiens was sprayed to run off with 105 condia/mL of Botrytis cinerea (strain IMI 169558 [76]) in potato dextrose broth. After a period of 1 h to allow air drying the infectedplants were placed in a 2 L capacity cuvette. Due to the requirements of the Sievers Nitric Oxide Analyzer [NOA 280i] chemiluminescent analyser that was used the flow ratewas set to 14 L/h. The airflow was divided to feed into the NOA280i and into QCL NO detector. Due to the constraints of the QCL a mass flow controller (MFC) limited the flowr 80i arm

aafppmamp

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ate to 1 L/h. As both signals are normalised to 1 L/h the signals from QCL and NOA 2easured using QCL and NOA 280i chemiluminescent systems.

ny possible contribution to the NO signal by water, ethylenend NO2 [67]. LAPD allowed us to measure NO productionrom tobacco and Arabidopsis plants challenged by bacterialathogens [12,67]. With a delay of only 2.5 min between inlanta emission and detection within the photoacoustic cell, theeasurements were near-contemporaneous and continuous. We

lso demonstrated a detection limit of around 20 pmol whichakes LAPD comparable to the chemiluminescence NO detection

latform [67].Although this represented a significant advance in plant NO

easurements there are considerable logistical problems associ-ted with LAPD. The requirement for physically large, specialisedquipment hardly makes the LAPD platform one that could beidely employed by many groups—unless the experiments were

ufficiently portable to allow measurements to be made at aingle place, for example the Trace Gas Facility at Radboud Uni-ersity (Nijmegen, the Netherlands). Furthermore, although LAPDs a sensitive technique it is unlikely to detect NO productionrom a smaller numbers of cells (for example, stomata) and nat-rally; given that NO is being detected in the gas phase, nopatial information can be deduced. Technical limitations centreound the need to ensure the removal of water vapour whose

hotoacoustic signal will interfere with the detection of a rangef trace gases. In Mur et al. [66] study, water was removed by

cold trap but it may be possible to use a calcium hydratelter.

e directly comparable. (B) NO production from B. cinerea infected tomato plants as

3.2.2. Quantum cascade lasers (QCL) based detectionThe recent advances of thermo-electrically cooled QCLs operat-

ing in the NO fundamental absorption band opened new avenueto the development of QCL-based NO detectors [68]. The bulkyCO lasers that need cryogenic cooling are nowadays replaced withQCLs. The advances of thermo-electrically cooled QCLs have createda range of novel mid-Infrared gas sensors. Operating at ambienttemperatures with high output powers and excellent spectral qual-ity, the QCL-based detector has opened new applications for gassensing due to its compact size, robust construction, high sensitiv-ity and low power requirements. Since few years, QCLs emitting inthe NO fundamental absorption band have trigger the developmentof QCL-based NO detectors.

We present a QCL used in combination with an a-stigmatic mul-tipass cell (effective path length 76 m) and wavelength modulationspectroscopy to detect NO at and below the part-per-billion byvolume (ppbv) mixing ratio [65]. A schematic arrangement of theexperimental setup is shown in Fig. 5. The QCL has a thermoelectri-cally cooled housing (Alpes Lasers). It operates in continuous waveat the wavelength region between 1847 and 1854 cm−1 whereNO shows strong absorption features, and at which the laser pro-duces an output power of 25 mW at 245 K. The laser beam passes

through a multipass absorption cell (Aerodyne, AMAC-76) andundergoes multiple reflections between two mirrors, thus enhanc-ing the light–NO molecule interaction. The difference in strengthbetween the light coming into the cell and the transmitted light

516 L.A.J. Mur et al. / Plant Science 181 (2011) 509– 519

Fig. 7. Reduced NO detection with the inclusion of non-infected plant tissue. (A) NO production from a 5-week-old (short day 8 h light) Arabidopsis Col-0 plant grown ona module of Levingtons M2 compost (approximately 27 cm3. Pictured; bar = 1 cm) and from the same soil from which the plant has been removed by cutting the stem atthe soil surface. The rosette was therefore left intact. Reapplication of the cut rosette to the soil surface reduced NO production. (B) NO production from tobacco (Nicotianatabacum cv. Samsun NN) leaves inoculated with (1 × 106 bacteria/mL 10 mM MgCl2) Pseudomonas syringae pv. phaseolicola strain 1448 either over the entire leaf (100%) or,∼75%, ∼50%, ∼25% of the leaf at 6 h after challenge. Results are given as mean (n = 3 ± SE) nmol/h/g fwt. Pictured are leaves either entirely infected (100%), or, ∼75%, ∼50%,∼25% inoculated with P. s. pv. phaseolicola at 24 h after challenge so that tissue collapse illustrates the extent of inoculation (Bar = 1 cm). Based on the mean levels of NOp uctiona ggestsu

(aom5

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roduction with 100% leaf inoculation it is possible to predict the “theoretical” prodctual NO production rates are considerably lower than the theoretical. Our data suninfected tissue.

after the cell) is proportional with the NO concentration. Thispproach allows a sensitivity of 0.8 ppbv in 1 s. The main advantagef this NO detection system is the possibility for long-term auto-ated operation and low ventilation rate over the sample (max.

L/h), which is needed in plant biology.In order to test the performance of QCL-based NO detection,

t is compared with the chemiluminescent NO detection platformFig. 6). To this aim, the tomato ABA mutant sitiens was spray-noculated with a 105 conidia/mL suspension of Botrytis cinereao run-off. The plants were allowed to air dry for 1 h after whichhey were placed within a 2 L volume cuvette. The chemilumines-ent platform required an input flow rate of the carrier air of ateast 12 L/h whilst the QCL was limited to 1 L/h. Thus, 14 L/h wasassed through the cuvette which was subsequently split into flowsf 13 L/h to the chemiluminescent platform, and via a mass flowontroller (MFC) to 1 L/h to the QCL-based system (Fig. 6A). It ismportant to note that, irrespective of the split flows, both plat-orms are measuring NO content from the same cuvette that areormalised to rates of production per litre. The detection of iden-ical NO levels, using the QCL-based and the chemiluminescentased system, demonstrated the validity of the first (Fig. 6B). Were currently preparing a detailed description of NO detection in B.inerea infected tomato (Solanum esculentum) using QCL (Sivaku-aran et al., in prep.).In considering measurements of gaseous NO from plants, we

ust take into account instances where there have been mis-atches between reported NO production as detected usingAF-dyes and the chemiluminescent approach [15,39]. Thus, there

s an apparent lack of detection of NO in cryptogein treated plants

levels with leaves which have been inoculated over lower % of their area. Note that that this is due to oxidation of NO produced by infected tissue by the surrounding

and cultures using chemiluminescent detection compared to theuse of DAF dyes and NO electrodes [15,58]. The difficulties of usingDAF dyes have been outlined above, but before using such con-siderations to dismiss data derived from these dyes it is worthconsidering the preponderance of data supporting the generatedNO. It should not be denied that DAF dyes can indeed measure NO,which is supported by the fact that large numbers of importantstudies each using different methods have noted NO generationduring the hypersensitive response (HR) [3,16,31,67,69] and alsocryptogein treatment [26,31,58]. Most compellingly, we shouldalso strongly consider independent indicators of NO generationduring the HR. Recently, many groups have been focusing on pro-tein S-nitrosylation and nitration during the plant defence againstpathogens [6,70], which, besides yielding fascinating observations,represents an independent validation that NO is produced. Addi-tionally an important point of the Vitecek et al. [26] study wasthat their detection of NO production from the gas phase of cryp-togein inoculated tobacco suspension cultures was based on flowrates of 2.4 L/h and also included a substantial “signal integrative”step as the azodye accumulated in the second trap (Fig. 1B). Thereader should also note that in our comparison between QCL andchemiluminescent platforms (Fig. 6A), the flow of 14 L/h representsa considerable dilution of the signal compared to our usual 1–1.5 L/h[67]. When using our usual flow rate we measured NO concentra-tions in the region of 800 ppbv (data not shown). This far exceeds

the levels we detect from a bacterially elicited HR in Arabidopsisor tobacco which have never exceeded 80 ppbv (data not shown).Thus, our measurement of HR would have been diluted to belowthe detection limits if we had used flow rates of 14 L/h. We are

L.A.J. Mur et al. / Plant Science 181 (2011) 509– 519 517

Fig. 8. Nitric oxide detection in mildew based barley using DAF-2DA dye and quantum cascade laser (QCL). (A) NO production over a 43 h period in 1-week-old barley(Hordeum vulgare cv. Pallas 01 [P01]) uninfected and infected seedlings. The light–dark periods are indicated. The infection protocol was as described in Prats et al. [30]. Note,the increases in NO in uninoculated controls (labelled “a” and “c” in A) which correlated with the periods where stomatal closure was being initiated (data not shown). Theincreased NO production at points “a” and “c” were significantly (P < 0.01) different to NO production in the middle of the light period (labelled “b” in A). (B) Autofluorescenceand fluorescence after DAF-2DA treatment of the same samples taken at 12 h after infection (hpi), i.e. during papillae formation and at 14 hpi when hypersensitive response(HR) is being initiated and after formation of the HR. Note that these events are occurring mostly in the epidermal cell layer. Comparing (A) and (B), note that the increasesi iated

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n NO production as detected using QCL correlated with increases in DAF-2DA assocot autofluorescence could be suppressed upon treatment with 250 �M cPTIO (see

ot suggesting that this invalidates the otherwise excellent chemi-uminescent system but that configurations that use lower flowates should be used or the integration period over which a signals collected should be increased.

When comparing QCL and chemiluminescent systems we made serendipitous observation which has bearing on gaseous NO mea-urements from the air (Fig. 7). When including the module withoil and Arabidopsis rosette in the cuvette, we detected more NOroduction when the plant was cut and removed than when it wasresent. When the excised plant was reapplied to the surface ofhe soil, NO production was again reduced (Fig. 7A). This suggestedhat soil microbes were a major source of NO. Whilst this couldave reflected a masking of the NO signal by a plant volatile(s) weave since associated this loss of NO with its oxidation by plantemoglobins ([Hb] Mur et al. paper submitted). This suggests thathen measuring NO care must be exerted to make sure that asuch of the plant material under assessment is producing NO;

therwise oxidation by Hb would reduce the gaseous “lost” NOignal. In our case we have been fortunate that we have alwayssed heavily-inoculated tobacco leaves [67], Arabidopsis rosettes12] or tomato seedlings (Fig. 6). We suggest that wherever pos-ible, experimenters seeking to measure NO from the gas phasehould maximize the proportion of plant material producing NO.

In passing, these observations have implications regarding the

ources of NO generation. Whilst NR [51], NOS-like enzymes71], polyamine reducing enzymes [72], hydroxylamine reducingnzyme [62] and chemical reduction of nitrate [73] are clearlyources of NO, a suppression in Hb expression would increase con-

fluorescence but not autofluorescence. Increases in fluorescence with DAF-2DA butet al. [30]).

centrations of in planta NO. We would predict that such could bemost relevant in roots (Hb will oxidize NO at very low partial pres-sures of O2) [74], or in leaves of low lying rosette-type plants suchas Arabidopsis. Equally, localised suppression of Hb could aid tofurther elevate NO concentration when generated at, for example,a HR.

4. NO measurements—some recommendations

Our consideration of the preferred methods used by plant sci-entists to detect NO has highlighted their power to provide somecutting edge insights into NO biology. Equally, it has revealedsome considerable problems with each technique—whether thisbe doubts as to their specificity, simple logistical costs or the inap-propriateness to a system under investigation.

Until other NO reporting fluorescent dye become available,we accept that it is unavoidable that DAF-based dyes will con-tinue to be used. The ability that DAF stains have to report NOgeneration within discrete cell types is currently unparalleled.Confirmation that NO is indeed being measured should followthe following steps. Firstly, non-DAF treated materials should beimaged to ascertain background fluorescence that should be quan-tified, and the increase on application of DAF should be expressed

as a factor of the background (see Prats et al. [30]). Next, attemptsshould be made to suppress the putative NO fluorescence sig-nal with cPTIO and mammalian NOS inhibitors, if desired. Shouldthese reduce the fluorescence signal, this should not be taken as

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18 L.A.J. Mur et al. / Plant

efinitive proof (for the reasons mentioned above) but clearly, nouppression would indicate that NO is not being measured. Ulti-ate confirmation can involve the independent measurement ofAF-2T using high-pressure liquid chromatography (HPLC; Kaiser,. [Wurzburg, Germany] pers. comm.). If well-characterised NOutants (see Vitecek et al. [26]) or transgenic Hb lines (for exam-

le, hmpX over-expressing lines [75]) are available, these should besed in preference to any other control. Alternatively, NO produc-ion should be measured using more than one technique. A goodxample of this approach is provided by Bright et al. [49] whereO production from rehydrated pollen was measured using DAFyes and EPR. Similarly, Besson-Bard [58] used their homemadeicroelectrode to confirm the production of NO from cryptogein

reated tobacco cell cultures where patterns of production closelyaralleled those detected using DAF dyes. This ethos also under-inned the approach of Planchet and Kaiser [39] who attemptedo compare NO signals from cryptogein treated plant tissues usinghe chemiluminensce approach and DAF-2DA dyes. An illustrationf this approach from our own data is given in Fig. 8. We haveeported NO generation from Blumeria graminis f. sp. hordei (pow-ery mildew) challenged Barley cultivar P-01 [30]. In this cultivar B.raminis elicits single epidermal cell death or forms cell wall papil-ae, both of which are associated with NO generation. Such veryubtle patterns of NO generations represent a challenge for a gasased NO detection system. However, NO emissions determinedsing QCL closely matched the patterns previously reported usingAF-2DA (Fig. 8).

Obviously, many groups do not have access to a chemilumi-escent NO detector, or such specialised equipment as an EPResonator or LAPD, QCL devices. In such circumstances, we wouldrge such groups to consider using NO electrodes [62] (where an inlanta measuring approach has been demonstrated [60]) or, use theelatively neglected (by plant scientists) Griess reagent assay. Vite-ek et al. [26] have demonstrated how this can be used to measureO from the gaseous phases and allowing accumulation of azodyever time (Fig. 1B). This dramatically increases the sensitivity of theystem by increasing the length of time over which the NO signalan be integrated.

In summary, we suggest that adoption of robust NO measure-ent approaches will assuage much of the controversy that is a

eature of much of plant NO research.

cknowledgements

We would like to thank many spirited discussions with Prof.erner Kaiser (Wurzburg, Germany) for inspiring many of the

oints raised in this commentary. Thanks to Jessica Gough (Aberys-wyth, UK) for critically reading this manuscript. The work of LMs supported by the BBSRC and the EU-FP6-Infrastructures-5 pro-ram; project FP6-026183 ‘Life Science Trace Gas Facility’. EP isunded by Spanish government AGL2010-15936/AGR.

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