Flooding induced emissions of volatile signallingcompounds in three tree species with differingwaterlogging tolerancepce_2166 1582..1594

LUCIAN COPOLOVICI & ÜLO NIINEMETS

Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, Kreutzwaldi 1,Tartu 51014, Estonia

ABSTRACT

To gain insight into variations in waterlogging responsive-ness, net assimilation rate, stomatal conductance, emissionsof isoprene and marker compounds of anoxic metabolismethanol and acetaldehyde, and stress marker compoundsnitric oxide (NO), volatile products of lipoxygenase (LOX)pathway and methanol were studied in seedlings of tem-perate deciduous tree species Alnus glutinosa, Populustremula and Quercus rubra (from highest to lowest water-logging tolerance) throughout sustained root zone water-logging of up to three weeks. In all species, waterlogginginitially resulted in reductions in net assimilation and sto-matal conductance and enhanced emissions of ethanol,acetaldehyde, NO, LOX products and methanol, followedby full or partial recovery depending on process andspecies. Strong negative correlations between gs and inter-nal NO concentration and NO flux, valid within and acrossspecies, were observed throughout the experiment. Iso-prene emission capacity was not related to waterloggingtolerance. Less waterlogging tolerant species had greaterreduction and smaller acclimation capacity in foliage physi-ological potentials, and larger emission bursts of volatilestress marker compounds. These data collectively provideencouraging evidence that emissions of volatile organicsand NO can be used as quantitative measures of stresstolerance and acclimation kinetics in temperate trees.

Key-words: Alnus glutinosa; Populus tremula; Quercusrubra; anoxia; green leaf volatiles; methanol; nitric oxide;photosynthesis; stomata; volatile organic compounds.

INTRODUCTION

Complete or partial soil waterlogging inhibiting carbon gainand growth and increasing the risk of premature plantdeath is deleterious to majority of terrestrial plants.Tempo-ral flooding in areas with flat topography and abundantand/or seasonally distributed precipitation is currently a

major plant stress factor in many temperate countries in theNorthern hemisphere. In addition, global change bringsabout modifications in the overall amount and temporaldistribution of precipitation, and this along with elevatedsea level further increases the flooding stress danger in theNorthern hemisphere temperate forests (Parry et al. 2007).

Gaseous diffusion is four orders of magnitude slower inwater than in air, and accordingly, soil water saturation hasmajor repercussions on gaseous diffusion (Mommer, Ped-ersen & Visser 2004). One of the main effects of the floodingis a lower oxygen availability in the submerged plant part,and developing anoxic conditions will lead to reduced ATPproduction and consequent inhibition of root metabolism.The soil flooding also modifies soil pH and soil redox poten-tial, further altering plant metabolism and growth (Pezeshki2001). Plants exhibit large variations in the acclimationpotential and tolerance to soil waterlogging, and the inter-specific variation in flooding resistance is responsible fordifferences in species abundance and distribution inflood-prone ecosystems worldwide (Jackson, Ishizawa & Ito2009).

Anoxic conditions elicit a plethora of physiological stressresponses including reductions in photosynthesis rates andstomatal conductance (e.g. Jackson et al. 2009), elicitation ofemissions of marker compounds of anaerobic metabolismethanol and acetaldehyde (Atkinson, Harrison-Murray &Taylor 2008; Rottenberger et al. 2008), and production of aseries of volatile stress marker compounds such as metha-nol (Holzinger et al. 2000), lipoxygenase (LOX) pathwayproducts (Agarwal & Grover 2006) and nitric oxide (NO)(Dat et al. 2004; Dordas et al. 2004). However, so far thecause–effect relationships in decline in photosyntheticrates and stomatal conductance are not fully understood(Jackson 2002; Ehlert et al. 2009), and the components offlooding-driven signalling pathway are still under extensiveinvestigation (Yordanova & Popova 2007; Sairam et al.2008; Ehlert et al. 2009). Furthermore, quantitative relation-ships between species waterlogging tolerance and immedi-ate stress responses have been developed in only a fewstudies (Pezeshki 1994; Ranney & Bir 1994; Rätsch &Haase 2007). However, such relationships are needed togain insight into forest ecosystem responses to flooding(Dat et al. 2004).

Correspondence: Ü. Niinemets. Fax: +372 7 313738; e-mail:ylo.niinemets@emu.ee

Plant, Cell and Environment (2010) 33, 1582–1594 doi: 10.1111/j.1365-3040.2010.02166.x

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To evaluate the variation in immediate and acclimationresponses to soil waterlogging, we studied foliage photosyn-thetic rates, stomatal conductance and volatile compoundssynthesized in tissues under anoxia (ethanol, acetaldehyde)and under stress (methanol, LOX pathway products), andNO emission in three temperate tree species of contrastingflooding tolerance. Among the studied species, Alnus gluti-nosa (L.) Gaertn. is a widespread species in flooded marginsof waterbodies, Populus tremula L. is a typical species inclay-rich heavy soils with flooding conditions common inspring and after heavy rains,and Quercus rubra L.is a speciescharacteristic to mesic well-drained forests. According to arecent compilation of waterlogging tolerance for Northernhemisphere temperate species [all species ranked from 1(lowest) to 5 (highest) flooding tolerance, Niinemets & Val-ladares 2006], A. glutinosa has the highest waterlogging tol-erance (ranking score 3.1), followed by P. tremula (2.1) andQ. rubra (1.1).We hypothesized that the degree of reductionin photosynthesis and stomatal openness decreases andthe production of alcoholic fermentation end-productsincreases with decreasing species waterlogging toleranceand that the acclimation during sustained waterlogging isgreater in species with higher waterlogging tolerance. Wefurther hypothesized that these differences in carbon assimi-lation and anoxic metabolism are quantitatively reflected inthe production of volatile stress marker compounds.

Apart from improving the quantitative understanding ofspecies waterlogging tolerance, the results of current studybear major significance for atmospheric chemistry. Volatilehydrocarbons produced by plants significantly alter atmo-spheric reactivity and together with NOX (NO and NO2)participate in ozone formation reactions in the atmosphere(Fall 2003 for a review). Thus, quantitative relationshipsbetween flooding and induction of volatile hydrocarbonemissions and NO are needed to simulate atmosphericreactivity in the boundary layer of flooded ecosystems. Inaddition to stress-elicited compounds, Q. rubra and P.tremula are strong constitutive isoprene emitters, while A.glutinosa is a non-emitter (e.g. Kesselmeier & Staudt 1999).So far, the role of isoprene emissions for plant performancehas not yet been fully elucidated, but there is evidence thatisoprene protects against a variety of stresses, possibly viaquenching stress-driven reactive oxygen species (ROS)(Vickers et al. 2009 for a review). Although reducing condi-tions prevail in the submerged root system, ROS formationis often enhanced in the leaves of flooded plants exposed tonormal concentrations of atmospheric oxygen (e.g. Visseret al. 2003; Yordanova, Christov & Popova 2004). On theother hand, isoprene emissions may itself be altered bystress effects. Isoprene emissions are known to be essen-tially insensitive to mild stress, and significantly decreaseunder strong environmental stress (Niinemets 2010 for areview) reflecting reductions in carbon availability for iso-prene synthesis. To our knowledge, flooding effects on iso-prene emission in species varying in flooding tolerance havenot been studied. We hypothesized that sustained floodingreduces isoprene emissions, and that this reduction isstronger in less tolerant species Q. rubra.

MATERIALS AND METHODS

Plant material

The plants of A. glutinosa and Q. rubra were grown fromseed (seed sources, Oudewater, the Netherlands, 52°01′N,4°52′E for A. glutinosa, and Ede, the Netherlands 52°02′ N,5°40′E for Q. rubra), while the plants of P. tremula weremicropropagated from clonal material of local origin(58°22′ N, 27°20′ E) (triploid aspen, Vardja, Tamm & Enno2004 for clonal propagation methods).After seedling estab-lishment, the plants were grown in 3 L clay pots filled witha mixture of commercial potting soil (Biolan Oy, Kauttua,Finland) and quartz sand (1:1) under incident quantum fluxdensity of 200 mmol m-2 s-1 (HPI-T Plus 400 W metal halidelamps, Philips, Hamburg, Germany) for 12 h photoperiodand day/night temperatures of 24/18 °C. The plants werewatered daily to field capacity with tap water and fertilizedonce per month with slow release NPK (3-1-2 ratio) fertil-izer with microelements. In all experiments, 6-month-oldtree seedlings of similar size were used. The seedlings ofsmaller-leaved species A. glutinosa (25 cm height) and P.tremula (30 cm height) possessed 10–15 leaves, while theseedlings of larger-leaved Q. rubra (30 cm height) had 6–8leaves at the time of the experiments. For all species, fourindependent flooding experiments were conducted andaverages were calculated.

Flooding experiments, and leaf area and drymass determinations

The plants were preconditioned at 350 mmol m-2 s-1 light(12 h photoperiod), at 30 °C/20 °C (day/night) temperatureand air humidity of 60% for 1 week before the start of theexperiments. The experimental root zone flooding treat-ments were always started in the morning just after the startof the light period. The treatments were started by placingthe pots with plants in polyethylene containers filled withdistilled water to a depth of 5–10 cm over the top of thepot edge.

The flooding treatment lasted until the stable values ofnet assimilation rate and stomatal conductance wereobserved but at least for 11 days following the day of thestart of the treatment (day 0) for all replicates per species(n = 4 for control and flooded plants). In A. glutinosa,where sustained high-level methanol emissions and in Q.rubra where sustained NO emissions were observed(see the Results), the experiment was continued for18–21 days.

After the flooding treatment, all leaves were harvested.Leaves were scanned at 300 dpi and projected leafarea was determined by custom-made software. Leaf drymass was estimated after drying the leaves for 48 h at70 °C. As the experiment was conducted in plantswith fully mature leaves and no new leaf growth wasobserved in both the control and flooded plants inany of the species, leaf area was stable throughout theexperiment.

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Gas-exchange system setup

An open two-channel gas-exchange system described indetail in Rasulov et al. (2009, Fig. 1 for the system setup)was used. In our setup, thermostatted 1.2 L glass chamberwas used, the gas flow rate through the system was1.4 L min-1, and CO2 and H2O exchange was monitoredwith an infrared dual-channel gas analyzer operated in dif-ferential mode (CIRAS II, PP-systems, Amesbury, MA,USA). NO concentrations at the chamber in- and outletswere measured with a Trace Level ChemiluminescenceNO-NO2-NOX Analyzer (model 42i-TL, Thermo-FischerScientific, Franklin, MA, USA) with a detection limit of ca.10 ppt, while volatile organic compound (VOC) concentra-tions were measured with a proton transfer reaction-massspectrometer (PTR-MS, high sensitivity version, IoniconGmbH, Innsbruck, Austria). Methanol, acetaldehyde,ethanol and isoprene were detected as protonated parentions at m/z of 33, 45, 47 and 69, respectively (Filella et al.2007 for details). The sum of volatile octadecanoid pathwayproducts (LOX products), consisting of a variety of C6

aldehydes produced from C13 hydroperoxides by hydrop-eroxide lyases and further rapidly metabolized to corre-sponding C6 alcohols and C8 esters (Feussner &Wasternack 2002), was found as the sum of the individualmass signals at m/z 83, m/z 85, m/z 99 and m/z 101 (Beau-champ et al. 2005; Velikova, Fares & Loreto 2008). Massspectra of pure chemicals (Sigma-Aldrich, St. Louis, MO,USA) were employed to verify the detection of differentLOX products by PTR-MS and gas chromatography-massspectrometry (QP2010 Plus, Shimadzu Corporation, Kyoto,Japan) equipped with a fused silica capillary columnZB-624 (Phenomenex, Torrance, CA, USA). The massspectra obtained confirmed the previous observations(Beauchamp et al. 2005; Velikova et al. 2008) that the pro-tonated mass fragment m/z 83 corresponds to hexenal andhexenol isomers and (Z)-3-hexenyl acetate, the protonatedmass fragment m/z 85 to 1-hexanol, the parent ion m/z 99 to(E)-2-hexenal and (Z)-3-hexenal and the parent ion m/z101 to (E)-2-hexenol and (Z)-3-hexenol. The PTR-MS wascalibrated using standard certified gas mixture (lot 3821,Ionimed Analytik, Innsbruck, Austria), consisting of 17 key

Figure 1. Schematic overview of the two-channel custom-designed gas-exchange system for measurements of the rates ofphotosynthesis, transpiration and trace gas emissions in plants. Ambient air is drawn from outside by a pump, purified by passing throughan ozone trap and charcoal filter, humidified to a desired water vapour concentration and passed through a buffer volume to even out thefluctuations in ambient CO2 concentrations. Flow rate through the chamber is fixed at 1.4 L min-1 by calibrated orifices D1 and D2. TheCO2 and water vapour concentrations are measured by infrared gas analyzer CIRAS II (PP-systems, Amesbury, MA, USA), while volatileorganic compound concentrations (VOC) are measured by high-sensitivity version of proton-transfer reaction mass spectrometer (PTRMS, Ionicon GmbH, Austria), and NO by a Trace Level Chemiluminescence NO-NO2-NOX Analyzer (model 42i-TL, Thermo-FischerScientific, Franklin, MA, USA). As CIRAS II is a true dual-channel analyzer, CO2 and H2O concentrations of incoming air arecontinuously measured in the reference channel. The electronic valves VM and VR are used to switch between the chamber and incomingair stream to measure NO and VOC interchangeably in ingoing and outgoing air, and match the CO2 and H2O gas-analyzers (when boththe reference and measurement channels measure the ingoing air).

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plant-emitted compounds at 1 ppm (�5%) range. In allexperiments, we used ambient air low in volatile organics(isoprene <1 ppb), ozone (<20 ppb) and NOX close to detec-tion limit (~0.01 ppb). The ambient air was further purifiedby passing through an ozone trap and charcoal filter, result-ing in very low ozone (1–2 ppb, and close to or below thedetection limit of VOC and NOX concentrations).

Gas-exchange measurements

For the gas-exchange measurements, the above-ground partof the plant was enclosed in the chamber, photosyntheticquantum flux density was set to 650 mmol m-2 s-1, air tem-perature to 30 °C (leaf temperature 29–31 °C) and relativehumidity 60%, and the CO2 concentration inside thechamber was ambient 380–400 mmol mol-1. All gasexchange rates were recorded after the steady-state valueswere reached, typically 30 min after the plant enclosure inthe chamber. Foliage CO2, water vapour and trace gasexchange measurements were conducted continuously forthe first three days of the experiment, and thereafter, onceper day between 11:00 and 14:00 h in both flooded andcontrol plants.

The net assimilation rate (A), transpiration rate (E), sto-matal conductance (gs) to water vapour and CO2 concen-tration in sub-stomatal cavities (Ci) were calculated fromthe measurements of CO2 and H2O concentrations atchamber in- and outlets according to von Caemmerer &Farquhar (1981). Analogously, NO, isoprene and LOXproduct flux rates were calculated (Beauchamp et al. 2005).All gas exchange rates were expressed per unit projectedleaf area of all leaves enclosed in the chamber.

Calculation of internal concentrations oftrace gases

The trace gas i, emission flux rate out of the leaf, Fi, isexpressed as:

Φ i s,i i,i a,ic= −( )g c (1)

where gs,i is the stomatal conductance to compound i(mol m-2 s-1) and ci,i is the internal concentration and ca,i isthe ambient concentration of this compound (mol mol-1).From this, the internal concentration is given as:

c c gi i a,i i s,i., = + Φ (2)

gs,i for a given compound can be calculated from stomatalconductance to water vapour, gs, as (Ball 1987; Niinemets &Reichstein 2003):

g g D Di s i H20,= (3)

where Di is the binary diffusion coefficient for the givencompound and DH2O is that for the water (m2 s-1). Diffusioncoefficients at given temperature for most trace gases werefrom Niinemets & Reichstein (2003). For the compoundsnot reported in their study, the binary diffusion coefficient

at a given temperature was calculated according toChapman and Enskog (Tucker & Nelken 1982; Niinemets& Reichstein 2003). For instance, for NO a diffusion coef-ficient of 2.25 ¥ 10-5 m2 s-1 was obtained. This value agreeswell with previous estimates used in simulating NO diffu-sion in biological systems (Velikova et al. 2008; Hemmings-son & Linnarsson 2009). Using this estimate, the finalequation for ci,NO becomes:

c c gi,NO a,NO NO s,= + 1 19. Φ (4)

where 1.19 is the ratio of water vapour and NO (DNO)diffusivities (DH2O/DNO) at 30 °C (see Eqn 3).

RESULTS

Changes in net assimilation rate and stomatalopenness during flooding

Net assimilation rate (A) and stomatal conductance towater vapour (gs) initially declined in all species (Fig. 2,Table 1). This response was rapid, starting within 2 h afterthe start of the flooding treatment, but the temporal kineticsof the reductions in A and gs varied between the species.After 24 h of flooding, net assimilation rate (A) was reducedby 26% in A. glutinosa, 65% in P. tremula and 62% in Q.rubra compared with the non-flooded plants (Fig. 2) andthis decline was further emphasized by day 5 (Fig. 2,Table 1). The changes in A and gs occurred in parallel suchthat the CO2 concentration in sub-stomatal cavities (Ci) thatis driven by the ratio A/gs (Ci = Ca-1.56A/gs, where Ca is theambient CO2 concentration) varied little throughout thetreatment and decreasing only by 7% in P. tremula, by 20%in A. glutinosa throughout the treatment (Fig. 2c, the nega-tive correlations between the day of sampling and Ci bothsignificant at P < 0.005). In contrast, in Q. rubra, whole time-dependent trend in Ci was not significant (P > 0.05).However, during the initial 5 days of experiment character-ized by massive simultaneous reductions in A and Ci

(Fig. 2a,b), the decline of ca. 5% was only significant inA. glutinosa (P < 0.05), and Q. rubra was even characterizedby a positive trend in Ci (Fig. 2c). Despite some negativetrend throughout the experiment, the change in Ci was thatsmall that it could impossibly have been responsible for themassive reductions in A observed in this study.

The degree of recovery of foliage photosynthetic func-tion also differed between the species. In A. glutinosa, A andgs had recovered to pre-stress values by day 7 (Fig. 2a,b). InP. tremula, A and gs reached to ca. 70% of the initial pre-stress value 6–7 days after the flooding and stabilized atthese levels (Table 1). In Q. rubra, there was a slow increasein A after days 9–10, but the overall recovery was only ca.5% throughout the treatment (Fig. 2a). The photosyntheticactivity achieved by the end of treatment (full recoveryin A. glutinosa, recovery to ca. 70% in P. tremula and toca. 30% in Q. rubra) was significantly different betweenthe species (P < 0.01, according to one-way analysis ofvariance).

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Flooding-driven emissions of products ofanaerobic metabolism

In all species, flooding of the root system induced emissionsof ethanol and acetaldehyde (Fig. 3), reflecting alcoholicfermentation in flooded plants. Significantly enhanced emis-sions were observed after 24 h of flooding (Fig. 3). In allcases, the ethanol emission rates (Fig. 3a) were larger thanacetaldehyde emissions (Fig. 3b).

The emission rates, the ethanol-to-acetaldehyde emissionratios and emission dynamics differed significantly amongthe species. Q. rubra exhibited by far the highest emissionrates (e.g. 19.6 � 4.7 nmol m-2 s-1 for ethanol), followedby A. glutinosa (5.3 � 0.8 nmol m-2 s-1) and P. tremula(4.7 � 0.4 nmol m-2 s-1). In A. glutinosa, the maximumethanol emission was observed 3–4 days after the flooding

(a)

(b)

(c)

mm

Figure 2. Changes in net assimilation rate (a) and stomatalconductance to water vapour (b) per unit leaf area, and inintercellular CO2 concentration (c) in seedlings of three Northernhemisphere temperate deciduous trees species Alnus glutinosa(�, most waterlogging tolerant of the studied species), Populustremula (�) and Quercus rubra (�, most intolerant) after thestart of the flooding treatment. The measurements wereconducted at a quantum flux density of 650 mmol m-2 s-1, airtemperature of 30 °C and at ambient CO2 concentrations of380 � 20 mmol mol-1. Each data point is the mean (�SE) of fourindependent replicate experiments. At day 0, the entire rootsystems were completely submerged and the treatment wascontinued until foliage net assimilation rates and stomatalconductances stabilized, but at least for 11 d following the day ofthe start of the treatments (day 0). Dashed lines in (a) denote thelevel of recovery reached by the end of the treatment.

Table 1. Temporal variation in average(� SE) net assimilation rate (A) andstomatal conductance to water vapour (gs,both in % of the values at day 0) in controland flooded plants of three Northernhemisphere temperate deciduous woodyplant species (Fig. 2 for the entire timeseries)

Species Variable

Day 5 Day 11

Control Flooding Control Flooding

Alnus glutinosa A 102.0 � 2.0a1 60 � 5b 100.0 � 4.0a 97.0 � 3.0a

gs 103.7 � 5.1a 59.1 � 3.5b 100.4 � 4.2a 92 � 7a

Populus tremula A 96.7 � 4.1a 59.5 � 3.3b 96 � 6a 62.8 � 4.1b

gs 100.1 � 6.4a 52.7 � 2.6b 99.8 � 4.3a 73 � 8b

Quercus rubra A 97.8 � 2.2a 26.9 � 1.1b 96.8 � 3.2a 25.8 � 2.2b

gs 100.2 � 1.6a 33.0 � 2.9b 100.0 � 1.3a 37 � 6b

1Means with the same letter are not significantly different between the control and floodedplants (P > 0.05 according to one way analysis of variance, n = 4 for both control and floodedplants).At day 0 (corresponding to 100%), no differences between the control plants and theplants subjected to root system submergence were found (P > 0.05 for all comparisons).

(a)

(b)

FF

Figure 3. Temporal variations in ethanol (Fethanol, a) andacetaldehyde (Facetaldehyde, b) emissions per unit leaf area in Alnusglutinosa (�), Populus tremula (�) and Quercus rubra (�) afterthe start of the root-zone flooding at day 0. Treatments and datapresentation as in Fig. 2. Arrows denote the peak emissions foreach species.

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treatment, while ethanol emissions peaked at day 5 in Q.rubra and P. tremula (Fig. 3a). In A. glutinosa and P. tremula,the peak acetaldehyde emissions were observed on the 4thday, and in Q. rubra on the 5th day (Fig. 3b).

Elicitation of stress marker compoundemissions by flooding

Flooding treatment resulted in significant emissions of avariety of volatile stress marker compounds. Emissions ofnitric oxide (FNO) increased until day 3–4 in Q. rubra, anduntil day 4–5 in A. glutinosa and P. tremula (Fig. 4a). Afterreaching the maximum value, FNO stabilized at this highlevel in Q. rubra, and decreased gradually in A. glutinosaand P. tremula (Fig. 4a). The emissions of the octadecanoidpathway products, LOX products (FLOX), peaked at day 4in A. glutinosa and in Q. rubra, and at day 5 in P. tremula(Fig. 4b). After reaching the initial maximum, FLOX

decreased initially in all species with a secondary increasebeing observed in Q. rubra and P. tremula (Fig. 4b). Metha-nol emissions peaked at day 3 in Q. rubra, at day 4 in P.tremula and at day 5 in A. glutinosa (Fig. 4c). After theinitial peak, methanol emissions decreased in Q. rubra andP. tremula, while the emissions stabilized at high level in A.glutinosa (Fig. 4c). The secondary increase of methanolemissions in A. glutinosa at day 7 (Fig. 4c) was linked to thestart of formation of lenticels and adventitious roots at thestem base (not quantified in this study). No adventitiousroots were formed in the other two species during theexperiment.

Overall, the most flooding intolerant species, Q. rubra hadthe highest NO and LOX emissions before the stress, andgreatest amplification of NO emissions in response to stressand maintenance of these high emissions throughout thestress (Fig. 4). This species also had the highest transientemissions of LOX products and methanol, while the mosttolerant species, A. glutinosa had intermediate NO andlowest LOX and methanol emissions during the initial peak,but the highest continuous methanol emissions after theinitial transient maximum (Fig. 4). As the result of differenttemporal variations, the correlations between stress markercompounds throughout the experiment differed among thespecies. FNO was positively correlated with FLOX in A. gluti-nosa and in P. tremula, and Fmethanol and FLOX were positivelycorrelated in P. tremula (Fig. 5). Negative x-intercept in FNO

versus FLOX in A. glutinosa demonstrated that in this species,the rise in FLOX occurred earlier than the increase in FNO. Incontrast, non-significant intercept in P. tremula suggestedthat both FNO and FLOX increased simultaneously duringstress in this species. In addition, FLOX and Fmethanol werepositively correlated in P. tremula (r = 0.77, P < 0.01). Noother correlations were observed among the stress markercompounds (Fig. 5, P > 0.17 for other combinations).

Stomatal conductance in relation to flux andinternal concentration of stress markers

For all data pooled, stomatal conductance to water vapourwas best correlated with FNO [r = -0.96 for gs versus

log(FNO), P < 0.001 for all data pooled] and with the inter-nal NO concentration (Ci,NO, Fig. 6), with a range of rbetween -0.82 and -0.90 and P between 0.011 and 0.0001for individual species relationships. According to the analy-sis of co-variance (separate slope model with interactionterm), the slope of gs versus log(Ci,NO) was moderatelysteeper for Q. rubra than for the other two species(P = 0.043), but the slopes of gs versus log(FNO) did notdiffer among the species (P > 0.2).

(a)

(b)

(c)

FF

F

Figure 4. Emission time-courses of nitric oxide, NO (FNO, a),volatile products of octadecanoid pathway, LOX (FLOX, b) andmethanol (Fmethanol, c) expressed per unit leaf area from theplants of Alnus glutinosa (�), Populus tremula (�) and Quercusrubra (�) after the start of root-zone flooding at day 0.Treatments and data presentation as in Fig. 2. Arrows highlightthe first maxima of emissions for each species.

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Analogous negative correlations were also observed withLOX products, for example, r = -0.74 for gs versuslog(FLOX) (P < 0.001), but the correlations with methanolemission were weak and even slightly positive for all datapooled r = 0.37 for gs versus log(Fmethanol) (P < 0.02). Only in

Populus, this later regression was negative (r = -0.92,P < 0.001), while not significant in the other species.

Reduction of constitutive isoprene emissionsduring flooding treatment

Isoprene emission rate was strongly reduced in response toflooding in Q. rubra, and the flooding-driven reduction inisoprene emission occurred simultaneously with overallreductions in foliage physiological activity, for example, withreductions in the net assimilation rate with no recovery bythe end of treatment (cf. Figs 2a & 7a). Thus, the rates ofisoprene emission and net assimilation were correlatedthroughout the flooding treatment in this species (Fig. 7b),with particularly strong correlation found for the initialperiod of the treatment when both the decline in A andisoprene emission rate was the strongest (e.g.r2 = 0.95 for thefirst 5 days of the treatment and r2 = 0.89 for the first 10 daysof the treatment). In contrast, in P. tremula, isoprene emis-sion rate exhibited only ca. 20% reduction during the first5 days of flooding (P < 0.005 for the time-dependentdecline), and then the emission rate recovered to ca. 90% ofthe pre-stress value (Fig. 7a). As isoprene emission ratechanged less than the net assimilation rate (Fig. 2a), netassimilation and isoprene emission rates were not correlatedthroughout the flooding treatment in this species (Fig. 7b).

Figure 5. Correlations of the emissionrates of the stress markers NO (a) andmethanol (b) with LOX products inAlnus glutinosa (�), Populus tremula (�)and Quercus rubra (�) throughout theflooding experiment. Treatments and datapresentation as in Fig. 2. Data were fittedby linear regressions.

(a) (b)

F

FF

F

FF

F F

Figure 6. Correlation between stomatal conductance to watervapour, gs, and NO concentration (ci,NO, log-scale) in sub-stomatalcavities in Alnus glutinosa (�), Populus tremula (�) and Quercusrubra (�). NO internal concentration was calculated by Eqn 4. gs

versus log(Ci,NO) relationship for all data pooled was fitted bylinear regression. Experimental treatment as in Fig. 2.

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DISCUSSION

Reductions in foliage photosynthetic rates andstomatal openness in response to flooding

Root-zone flooding driven reductions in net assimilationrate (A) and stomatal conductance (gs, Fig. 2a,b) have beenobserved in numerous species (Kozlowski & Pallardy 2002for a review).As the reductions in gs curb CO2 entry into theleaves, and thereby can limit CO2 availability for photosyn-thesis, lower gs has been suggested to be directly responsiblefor lower A (e.g.Yordanova, Uzunova & Popova 2005).Thissuggestion has been partly confirmed by lower intercellularCO2 concentrations (Ci) in flooded plants (e.g. Yordanovaet al. 2005). However, in other studies, Ci was relativelyconstant (Bradford 1983; García-Sánchez et al. 2007; Yor-danova & Popova 2007; Else et al. 2009), or even increasedsomewhat under flooding (García-Sánchez et al. 2007; Tet-sushi & Karim 2007), suggesting that the reductions in gs

were not immediately responsible for the reductions in A.In our study, only minor changes in Ci were observed(Fig. 2c), indicating that A and gs declined in coordinatedmanner throughout the flooding treatment. Inhibition ofassimilate retranslocation from leaves to roots by anoxia(Saglio 1985) as is common under stress (Sowinski 1999),and resultant accumulation of sugars and starch in theleaves (Wample & Davis 1983; García-Sánchez et al.2007) have been shown to lead to feedback inhibition ofphotosynthesis in the flooded plants (Bradford 1983; Vu &Yelenosky 1991).

Given that the photosynthetic decline was not causedby changes in stomatal conductance, the decline in gs ispuzzling. Simultaneous reductions in photosynthesis andstomatal conductance have been observed for severalstresses (Wong, Cowan & Farquhar 1979), but suchco-variations in photosynthesis and stomatal openness arestill not fully understood. In the case of flooding, a con-comitant reduction in gs matching the decline in photosyn-thesis can be achieved directly by reduced root hydraulicconductivity because of closure of aquaporin water chan-nels in response to pH signals (Jackson 2002; Ehlert et al.2009) or by hormonal interactions such as the rise ofabscisic acid (ABA) concentration in the leaves becauseof inhibited export from the leaves, and as the result of

ABA – ethylene interaction (Else et al. 2009). In addition,elevation of ROS and NO concentrations is needed forABA-promoted stomatal closure (García-Mata & Lamat-tina 2003; Desikan et al. 2004; Wang & Song 2008; Wilson,Neill & Hancock 2008).

In our study, we found spectacular general negative cor-relations between the internal NO concentration and gs

(Fig. 6) and NO flux and gs, and it is tempting to speculatethat anoxia-induced elevation in NO production played arole in reductions in stomatal openness (see also thesection Production of stress hormone NO). However, areduction in gs inevitably results in build-up of NO insidethe leaves, and it may be argued that the correlationbetween internal NO concentration and gs simply resultsfrom changes in gs (Velikova et al. 2008). Yet, this argu-ment cannot be applied to explain the correlation of gs

with NO flux. NO has a very large gas–liquid phasepartition coefficient (Henry’s law constant) of56 290 Pa m3 mol-1 (Sander 2001), and is therefore prefer-ably distributed in the gas phase. Thus, its flux cannot beeffectively controlled by stomata as the reduction of theconductance is compensated by enhanced diffusion gradi-ent between the ambient air and leaf interior because ofthe rapid rise of internal gas-phase NO concentration(Niinemets & Reichstein 2003 for a quantitative modelanalysis). It follows that the negative correlation betweenthe flux of NO and gs points to NO controls on gs ratherthan gs controls on NO flux. Analogous correlationsbetween the internal NO concentration, NO flux and sto-matal conductance have been found by manipulating leafoxidative status and NO emission using artificial toxins inPopulus nigra (Velikova et al. 2008). To our knowledge,this is one of the first reports showing such a general cor-relation valid within the studied species as well as across arange of species subject to waterlogging treatment.

In our study, the seedlings of all species were also rela-tively tolerant to flooding and all survived the entire experi-ment. Nevertheless, species clearly differed in the degree offlooding-driven reductions in foliage net assimilation rateand stomatal conductance and the degree of recoveryduring flooding with A. glutinosa showing the smallestdepression and greatest recovery and Q. rubra thelargest depression and the most modest recovery (Fig. 2,Table 1).

Figure 7. Changes in isoprene emissionrate per unit leaf area during floodingtreatment (Fisoprene, a) and the correlationof Fisoprene with net assimilation rate (b) inPopulus tremula (�) and Quercus rubra(�). Treatments and data presentation asin Fig. 2. Data in (b) were fitted by linearregressions. The non-significant regressionis shown by a dashed line. Alnus glutinosaalso investigated in this study is not anisoprene emitter.

(a) (b)

m

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Flooding-induced emissions of markercompounds of anaerobic metabolism

In all species, flooding resulted in enhanced emissionsof ethanol and acetaldehyde from the leaves (Fig. 3), indi-cating engagement of alcoholic sugar fermentation underlimited oxygen availability as reported in previous studies(e.g. Kreuzwieser, Scheerer & Rennenberg 1999; Kolb &Joly 2009). Several studies have demonstrated that foliage-emitted ethanol originates from roots (Kreuzwieser et al.1999), while most of the acetaldehyde emitted from floodedtrees is formed from ethanol by alcohol dehydrogenase(Kreuzwieser et al. 1999; Kreuzwieser et al. 2001). Alcoholdehydrogenase activity is constitutively present in leaves,stems and roots (Kreuzwieser et al. 1999), and is signifi-cantly increased in roots after flooding (Kreuzwieser et al.1999). Nevertheless, acetaldehyde emission flux was rela-tively small compared with ethanol flux (cf. Fig. 3a,b). Infact, relatively volatile acetaldehyde is further converted tomuch less volatile acetic acid by aldehyde dehydrogenase(Niinemets & Reichstein 2003 for compound differences involatility), and enters into general metabolism.

Although these patterns seen in our study qualitativelyagree with previous observations, studies investigatingthe response of plants to flooding have not been routinelyconducted in multiple species. It has been suggested thatthe flooding tolerance is associated with higher alcoholdehydrogenase activity (Kreuzwieser, Papadopoulou &Rennenberg 2004; De Carvalho et al. 2008). For instance,among the European temperate trees, highest acetaldehydeemissions after flooding were found in most flooding toler-ant P. tremula ¥ Populus alba hybrid (Kreuzwieser et al.2004). However, in our study, Q. rubra, the least tolerantspecies, exhibited highest ethanol and acetaldehyde emis-sions, suggesting that the emissions are driven both by therate of ethanol formation as well as biochemical processesdownstream alcohol dehydrogenase. Although the capacityfor acetaldehyde formation was high in Q. rubra, matchingcapacity for its metabolization was lacking in this species.

Production of stress hormone NO duringflooding treatment

It is now well recognized that NO is a component of therepertoire of signals used by plants (for reviews see Neill,Desikan & Hancock 2003; Wendehenne, Durner & Klessig2004; Wilson et al. 2008), and the formation of the signaltransduction molecule NO in response to various environ-mental stresses has been frequently observed (Desikanet al. 2004; Dordas et al. 2004; Besson-Bard, Pugin & Wen-dehenne 2008).The sources of NO in plants are not entirelyknown, but formation from nitrate by nitrate reductase hasbeen suggested as the major NO synthesis pathway (Wilsonet al. 2008). Under anoxic conditions, changes in pH andredox status are supposed to promote nitrate reductaseactivity, resulting in enhanced NO emissions (Dordas et al.2004). NO produced during stress is apparently involved inanoxia-dependent stomatal closure (s. above) and may be

involved in aerenchyma and adventitious root formationtogether with ethylene (Igamberdiev et al. 2005; Sairamet al. 2008). In addition, anoxia induces production of hae-moglobins that may oxidize NO and NADH and it has beenhypothesized that this reaction contributes to maintenanceof plant redox and energy status under hypoxia (Igamber-diev et al. 2005).

As our study demonstrated, species with varying floodingtolerance largely varied in NO production under floodingstress with NO production in Q. rubra, the most intolerantspecies, being significantly larger than in the other twospecies (Fig. 4a). Although the peak NO emissions werelarger in A. glutinosa than in P. tremula, the NO emissionsrecovered to a lower value in A. glutinosa. Thus, NOemissions during longer-term flooding matched the speciesranking according to waterlogging tolerance.

Emissions of other volatile stress markercompounds from waterlogged plants

LOX pathway products are induced in a variety of plantspecies during stress conditions. These compounds areformed from free octadecanoid fatty acids (linoleic acid,18:2 and linolenic acid, 18:3) released from plant mem-branes by phospholipases upon oxidative stress (Feussner& Wasternack 2002; Matsui 2006). Constitutive and stress-elicited LOX activity further results in formation of 9- or13-hydroperoxylinoleic or -linolenic acid or a mixture ofboth, and finally, as the result of hydroperoxide lyase activ-ity and further dehydrogenase reactions, various C6 alde-hydes and alcohols are produced (Feussner & Wasternack2002; Matsui 2006). Thus, the emission of LOX pathwayproducts is a reliable marker of oxidative stress (Feussner &Wasternack 2002; Porta & Rocha-Sosa 2002). The emissionof LOX products is elicited in response to many stressfactors (Heiden et al. 2003) and the sum of LOX productemission has been shown to scale with the severity of thestress (Beauchamp et al. 2005; Capitani et al. 2009).

In our study, enhancement of LOX product emissionswas found in all species during the first days of flooding andthis initial enhancement was correlated with increases inNO emissions, except in Q. rubra, where initial level of LOXemission was larger than in the other two species (cf. Figs 4& 5). This result is consistent with the observations thatoxidative stress and electrolyte leakage are induced byflooding (Yordanova & Popova 2007).

LOX product emissions during waterlogging reached thepre-stress values in only the most flooding-resistant speciesA. glutinosa and remained at high level in the other species(Fig. 4), with species final LOX emission rates again agree-ing with the waterlogging tolerance ranking.

Methanol emissions in relation to flooding andevidence of acclimation

In addition to LOX product emissions, many stress factorsalso elicit significant methanol emissions from plants

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(Beauchamp et al. 2005; Peñuelas et al. 2005; Cojocariu et al.2006). These emissions are associated with the activationof pectin methylesterases and demethylation of cell wallpectins (Micheli 2001; Pelloux, Rustérucci & Mellerowicz2007). In all species, methanol emissions initially increasedin response to developing anoxia, agreeing with the under-standing that activation of pectin methylesterases is one ofthe first steps of stress-dependent oxidative signalling.

Apart from stress-dependent emissions, large methanolemissions are found in growing tissues (Harley et al. 2007;Hüve et al. 2007). These emissions are also associated withrepeated methylation and demethylation of pectins, rigidi-fying and softening the walls of growing cells (Fall &Benson 1996). Typically, sustained waterlogging leadsto adventitious root formation in waterlogged plants(Mommer et al. 2004; Tetsushi & Karim 2007; Else et al.2009), improving oxygen availability in root system(Mommer et al. 2004). Elicitation of such a growth activitycan enhance methanol emissions. In addition, lysogenicactivity as the result of aerenchyma formation in floodedplants (Drew, He & Morgan 2000; Jackson et al. 2009) andresultant activity of hydrolytic enzymes in cell walls(Bragina, Rodionova & Grinieva 2003), possibly includingpectin methylesterases, can also result in methanol emis-sions from degraded pectins. In our study, only in A. gluti-nosa, formation of lenticels and adventitious roots wasobserved, although not quantified, and it is tempting tospeculate that the elevated methanol emissions found after7 d flooding in this species (Fig. 4c) are associated withadventitious root formation as found in other flooding-tolerant Alnus (Yamamoto, Sakata & Terazawa 1995;Iwanaga & Yamamoto 2007) species.

In A. glutinosa where extensive adventitious root for-mation was observed, foliage photosynthetic rates and sto-matal conductances fully recovered to pre-stress values,indicating full acclimation (Fig. 2). Analogous recoveryhas been observed in seedlings of other woody speciesforming adventitious roots (Iwanaga & Yamamoto 2007).The photosynthetic recovery was further associated withreduced ethanol and acetaldehyde formation, suggestingimproved oxygen availability in the root zone. In water-logged seedlings of Tabebuia, the reduction of alcoholdehydrogenase activity has been observed simultaneouslywith aerenchyma formation (Kolb & Joly 2009). Differ-ently from Alnus, only partial photosynthetic acclimationwas observed in P. tremula and essentially no acclimationin Q. rubra (Fig. 2), although the alcoholic fermentationreached to pre-stress level (Fig. 3). At the same time, NOemission in Q. rubra and LOX product emissions in bothspecies remained at high level, indicating chronic stress inthese species.

Flooding effects on constitutiveisoprene emissions

As noted in the Introduction, isoprene emission rate is rela-tively insensitive to moderate environmental stress, butacute stress typically leads to large reductions in isoprene

emission rate. In our study, isoprene emission rate declinedstrongly in Q. rubra throughout the waterlogging treatment(Fig. 7a) and this reduction paralleled the decreases in netassimilation rate, especially at the beginning of the treat-ment (Figs 2a & 7b). Analogous reductions in isopreneemission after flooding have been observed in several Ama-zonian species (Bracho Nunez et al. 2009). In contrast, onlymoderate initial decline and almost full recovery of iso-prene emission rate was observed in the more tolerantspecies P. tremula (Fig. 7b). As the result of more resistantisoprene emission than net assimilation, isoprene emissionrate was uncoupled from the rate of net assimilation in thisspecies (Fig. 7b). Such uncoupling has been observed pre-viously in the case of several other stress factors (Fares et al.2006; Brilli et al. 2007).

As discussed in the Introduction, volatile isoprenoids areconsidered to protect against a broad array of stressesleading to ROS formation. ROS formation in leaves withflooded root systems has been shown to play a role inflooding-driven decline in plant physiological activity(Visser et al. 2003; Yordanova et al. 2004), and enhancedLOX product emissions were observed in our study, consis-tent with greater oxidative stress in the leaves of floodedplants. Thus, isoprene could play an important role inquenching ROS in flooded plants. However, our study dem-onstrates that the capacity for isoprene emission was notlinked to flooding tolerance in the three species studied.Isoprene emission was lacking in the most flooding-tolerantspecies A. glutinosa, while the species with highest isopreneemission rate, Q. rubra, was most intolerant of floodingand could not sustain high isoprene emission rates underflooding.

CONCLUSIONS

Overall, the presented data provide exciting evidence thatthe species acclimation potential matched the waterloggingtolerance ranking, and that the VOC and NO emission mea-surements together with ethanol and acetaldehyde emis-sions can provide quantitative insight into the differences inspecies constitutive waterlogging tolerance and into time-courses of species waterlogging stress development, andacclimation to stress. Albeit these results are highly encour-aging, more experimental work including more species isneeded to test the generality of the observed patterns,and to directly test the hypothesis about the linkage ofstress marker compounds and changes in plant structure(e.g. adventitious root formation in relation to methanolemission).

ACKNOWLEDGMENTS

This work was supported by the Estonian Ministry ofScience and Education (grant SF1090065s07) and EstonianScience Foundation (post-doctoral grant JD101 and grant7645). We especially thank Kaia Kask for the skilledtechnical assistance.

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Received 11 February 2010; accepted for publication 26 March 2010

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