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Page 1: Tolerance of peat-grown Scots pine seedlings to waterlogging and drought: Morphological, physiological, and metabolic responses to stress

Forest Ecology and Management 307 (2013) 43–53

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Forest Ecology and Management

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Tolerance of peat-grown Scots pine seedlings to waterloggingand drought: Morphological, physiological, and metabolic responsesto stress

0378-1127/$ - see front matter � 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.foreco.2013.07.007

⇑ Corresponding author. Tel.: +358 40 801 5548.E-mail addresses: [email protected] (M. Pearson), markku.saarinen@

metla.fi (M. Saarinen), [email protected] (L. Nummelin), [email protected] (J. Heiskanen), [email protected] (Tytti Sarjala), [email protected] (J. Laine).

1 Professor emeritus.

Meeri Pearson a,⇑, Markku Saarinen a, Laura Nummelin b, Juha Heiskanen c, Marja Roitto d,Tytti Sarjala a, Jukka Laine a,1

a Finnish Forest Research Institute, Western Finland Regional Unit, Kaironiementie 15, 39700 Parkano, Finlandb University of Helsinki, Department of Forest Sciences, P.O. Box 27, 00014 Helsinki, Finlandc Finnish Forest Research Institute, Eastern Finland Regional Unit, Juntintie 154, 77600 Suonenjoki, Finlandd Finnish Forest Research Institute, Eastern Finland Regional Unit, P.O. Box 68, 80101 Joensuu, Finland

a r t i c l e i n f o a b s t r a c t

Article history:Received 19 March 2013Received in revised form 7 June 2013Accepted 5 July 2013Available online 31 July 2013

Keywords:Scots pine seedlingsPeatDroughtWaterloggingTolerance

Depending on the soil preparation method applied and peat characteristics, Scots pine (Pinus sylvestris L.)seedlings planted in prepared spots on forestry-drained peatlands may become more susceptible toextreme weather events such as drought or flooding. Only by studying its coping strategies can we even-tually design methodology better suited for Scots pine regeneration on peat soils. In this study, we eval-uated the tolerance of two-year-old seedlings to the two extremes of water-associated stress, droughtand waterlogging, in unprocessed peat. Over one growing season in controlled conditions, drought dis-tinctly reduced root and shoot growth in addition to photochemical efficiency (Fv/Fm) particularly in pre-vious-year needles, whilst wet stress had little discernible impact. Drought also influenced polyaminemetabolism by increasing free putrescine and spermine concentrations especially in current-year nee-dles, whereas no impact was discerned in the wet treatment. Furthermore, reduced root hydraulic con-ductance (Kr) was observed in drought-stressed root systems. Apparently, waterlogging does not modifyScots pine seedling growth or vitality immediately, but rather in the longer term. However, no fatalitiesoccurred in either of the stress treatments, this despite water availability in the Sphagnum peat reachingits lower (permanent wilting point) and upper (10% soil air content) limits. Maintenance of rather highphotochemical efficiency despite severe drought stress would seem to indicate a potential for seedlingrecovery if water availability in the peat substrate improved.

� 2013 Elsevier B.V. All rights reserved.

1. Introduction the stress, plant species as well as its developmental stage (Islam

In trees, water stress manifests as visible change such as leafyellowing or wilting, retarded growth, or disturbance to physiolog-ical processes (e.g., photosynthesis and root hydraulic conductiv-ity) (Kozlowski et al., 1991; Martínez-Vilalta et al., 2004; Thapaet al., 2011). Just as too little soil water poses a health risk to newlyplanted seedlings, too much water can be equally as debilitating.Oddly enough, both soil drying and flooding can lead to leaf dehy-dration (Aroca et al., 2012; Kramer and Boyer, 1995). The impact ofdrought or flooding on plant function will however depend on theduration, intensity and timing (e.g., dormant vs. growing season) of

et al., 2003; Kozlowski, 1984; Martínez-Vilalta et al., 2004).On forestry-drained boreal peatlands, one of the primary aims

of soil preparation is to manipulate soil water conditions for thebenefit of young tree seedlings. Typically, the focus has been ondraining surplus water from the peat to improve aeration ratherthan water retention. This emphasis, however, may change in thefuture as new climatic scenarios unfold. For instance, the frequencyof drought is predicted to increase in the boreal forest zone (IPCC,2007). Depending on the soil preparation method employed andpeat characteristics, seedlings may experience heightened suscep-tibility to desiccation (Saarinen, 1997, 2005) or alternatively toflooding (Pearson et al., 2011) in the face of extreme weather.Fickle summertime weather patterns already complicate regenera-tion efforts; an elevated peat mound may provide ideal substratefor seedling establishment one year, but be far from it the next(Saarinen, 2005). Thus, both now and in the future, successfulregeneration rides on the capability of seedlings to deal with waterstress.

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44 M. Pearson et al. / Forest Ecology and Management 307 (2013) 43–53

Scots pine (Pinus sylvestris L.) is a versatile conifer inhabitingboth dry and wet environments from sandy upland soils to water-logged organic ones. In Finland, the majority of forestry-drainedpeatlands are dominated by Scots pine. Thus, from forestry as wellas ecological standpoints, it is a key species. While some of themechanisms Scots pine employs for dealing with extremely dryor wet conditions during the growing season may be similar, thereis also evidence of differing mechanisms of adaptation. For in-stance, while drought has been shown to clearly reduce seedlingroot and shoot growth (Otronen and Rosenlund, 2001; Pearsonet al., 2013; Rikala and Puttonen, 1988), Scots pine seedlings sub-jected to flooding have demonstrated considerable tolerance withlittle or no impact on growth at least in the short term (Mukassabiet al., 2012; Otronen and Rosenlund, 2001; Zaerr, 1983). Scots pineis also known to employ a strategy of early stomatal closure to lim-it water stress (Picon-Cochard et al., 2006), which consequentlylowers transpiration, respiration, as well as net carbon assimilation(Bréda et al., 2006; Matyssek et al., 2006; Panek, 2004; Sterck et al.,2008; Zweifel et al., 2005, 2007). Furthermore, the phenologicalbehavior of the plant species cannot be ignored when studyingits response to stress. Several studies have shown that root growthis depressed during shoot and needle elongation in young seed-lings of Scots pine (Iivonen et al., 2001; Mattsson, 1986) and othernorthern pines, lodgepole pine (Pinus contorta Dougl. ex Loud.)(Cannell and Willet, 1976) and red pine (Pinus resinosa Ait.) (Drew,1982). Since growing shoots are strong consumers of assimilates inScots pine (Iivonen et al., 2001; Lyr and Hoffmann, 1967), rootgrowth tends to be most intense towards the end of the growingseason when shoot growth is nearly complete (Iivonen et al., 2001).

Chlorophyll fluorescence is indicative of the state of Photosys-tem II (PSII) and thus photochemical efficiency (Maxwell and John-son, 2000). As a technique, it is useful in determining the ability ofa plant to tolerate stress as well as the extent of damage incurredby the photosynthetic apparatus in response to stress (e.g.,Mohammed et al., 1995; Maxwell and Johnson 2000). In describingthe photochemical efficiency of PSII, an Fv/Fm value of approxi-mately 0.83 is generally considered normal for healthy plants,and a sustained decline in Fv/Fm is indicative of plant stress (Max-well and Johnson, 2000). With regards to Scots pine, chlorophyllfluorescence has typically been used as an indicator of other typesof stress instead of drought or waterlogging (e.g., Gielen et al.,2000; Taulavuori et al., 2000).

Root hydraulic conductance (Kr) and conductivity (Lp) indicatethe water uptake capability of roots. Roots with high hydraulicconductance are less resistant (i.e., more permeable) to water flowthan those with low hydraulic conductance (Aroca et al., 2012).Typically, root resistance to water flow increases in response tostress—e.g., drought or poor root aeration due to waterloggedsoil—consequently leading to reduced root hydraulic conductance(e.g., loblolly pine (Pinus taeda L.), Lee et al., 1990); tamarack (Larixlaricina (Du Roi) K. Koch) and black spruce (Picea mariana (Mill.)B.S.P.), Islam et al., 2003; Islam and MacDonald, 2004; southernpines, Sword Sayer et al., 2005). Increased root lignification andsuberization have been implicated in decreased root permeabilityand hydraulic conductance (Lo Gullo et al., 1998; North and Nobel,1991; Sands et al., 1982; Trifilo et al., 2004). Within the Pinaceaefamily, considerable variability in the effects of flooding stress onroot hydraulic conductance and conductivity and seedling vitalityand growth, in general, is evident (Kozlowski, 1997).

Polyamines are ubiquitous biogenic amines involved with dif-ferent types of abiotic and biotic stresses (Alcázar et al., 2010),and also play a well-established role in most cellular processesduring growth and development of plants (Hussain et al., 2011;Moschou et al., 2012). The polyamines spermidine, spermine, andtheir precursor putrescine are the most common and thus alsothe most frequently studied, and have been reported to serve as

intracellular messengers for conveying physiological responses(Davies, 2004). Notably, polyamine concentrations are affected bydrought stress in some plant species (Capell et al., 2004; Kasukabeet al., 2004; Ma et al., 2005) including young Scots pine (Pearsonet al., 2013). Tolerance to drought has also been associated withpolyamine biosynthesis for example in eastern white pine (Pinusstrobus L.) (Tang et al., 2007). The relationship between waterlog-ging stress and polyamine metabolism in plants has been muchless studied, but for instance exogenous application of putrescinehas been shown to increase plant tolerance to waterlogging in cropplants (Gill and Tuteja, 2010; Yiu et al., 2009).

Only by studying its coping strategies can we eventually designmethodology better suited for Scots pine regeneration on peatsoils. Hence, our objective was to evaluate the tolerance of plantedScots pine seedlings to the two extremes of water-associatedstress, drought and waterlogging, in unprocessed peat. As indica-tors of seedling vitality, we used chlorophyll fluorescence—a mea-sure of PSII photochemical efficiency—to recognize the earlyphysiological signs of stress, and endogenous polyamine concen-trations and root hydraulic conductance to describe the internaloutcomes of exposure to desiccated and waterlogged soils. In addi-tion, several shoot and root-related, post-treatment morphologicaltraits were recorded to determine the impacts on growth.

2. Materials and methods

2.1. Experimental design

The following experiment was carried out at the Finnish ForestResearch Institute in Parkano, Finland (62�0003500N, 23�0103000E)from late May to early September 2009. As part of a larger studydealing with soil preparation and forest regeneration on drainedpeatlands (Pearson et al., 2011), the substrate and seedling mate-rial selected for this experiment were typical for peatland forestry.The 99% organic substrate was dug up manually from a dwarfshrub type (Vatkg) of drained peatland (for classification seeVasander and Laine, 2008). To disengage the substrate in blockswithout disturbing its inner structure, the humus layer wasstripped off from an approximately 4 m2 area and 75 individualPVC frames (25 cm long � 20 cm wide � 20 cm high) shoved intothe peat. Thereafter, the delimited peat blocks were detached frombelow with a peat spade. These blocks consisted primarily ofpoorly decomposed Sphagnum peat (‘‘bottom’’), but were toppedby an approximately 5-cm-thick layer of Sphagnum peat, whichhad decomposed moderately in response to drainage (‘‘surface’’).

Each of the 75 blocks was planted with one, two-year-old con-tainerized Scots pine seedling on May 25th 2009. The stock wasfrom cold storage and the pots 72 cm3 in size (8 cm deep). Twoweeks later on June 7th, the seedlings were then randomly as-signed to control (CT), wet (WT), and drought (DT) treatments.One-third of the seedlings represented the control treatment andwere set in a PVC tub and watered from below. The water levelin the control was maintained at 18 cm below the top surface ofthe 20-cm-high blocks in order to ensure sufficient capillary watermovement and thus adequate moisture conditions throughout. Inthe drought treatment, 25 seedlings were placed atop planks andsubjected to drought by withholding water entirely. To speed updrying of the substrate, the PVC frames were removed from theseblocks. Each block was then tightly wrapped with chicken wire(1.3 cm diameter) to keep it together in case of crumbling as thepeat dried. In the wet treatment, 25 of the peat blocks planted withseedlings were put in a PVC tub and exposed to an elevated waterlevel for the duration of the experiment. The water level was con-sistently kept at 6–7 cm below the top surface of the peat blocks,which meant the seedling pots were about halfway submerged.

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M. Pearson et al. / Forest Ecology and Management 307 (2013) 43–53 45

The water level in the tub was monitored twice daily and wateradded to the bath as necessary. The seedlings were never wateredfrom above (i.e., on top of the soil). Throughout the experiment, allthe seedlings were located outside in a lean-to with a transparentroof allowing for 8 h of direct sunlight and shelter from the rain.Inflicting drought and wet stress under controlled conditions hasthe advantage of shutting out biotic interference (e.g., insect oranimal damage), which so often hinders interpretation of resultsin field studies. The treatments began on June 7th and ended onSeptember 7th 2009.

At the conclusion of the experiment, water retention character-istics of the substrate at desorption (relative to wet volume at�0.3 kPa) were determined separately for the surface and bottomSphagnum peat components of the blocks from five saturated,undisturbed fresh samples using a pressure plate apparatus (Soil-moisture Equipment Corp., USA) and procedures described in de-tail by Heiskanen (1993) at the Suonenjoki unit of the FinnishForest Research Institute (Fig. 1). At the same time, bulk density(g cm�3, dry mass to wet volume at �0.3 kPa) and total porosity(%, by volume in relation to wet volume at �0.3 kPa) were alsodetermined. The water retention curve indicates the level of suc-tion required by the plant to draw water from the soil as it dries.During the drying process, soil water increasingly moves from cap-illary pore space and adheres to soil particles, which consequentlyslows down plant water uptake (Päivänen, 1973). Respective bulkdensities were 0.07 and 0.10 g cm�3 and total porosities 96% and94% for the bottom and surface peats. The permanent wilting point(�1554 kPa, i.e., water no longer available to plants) (Taiz and Zei-ger, 1991) lies at approximately 10% and 12% soil water content(WC) for the bottom and surface peats, respectively (Fig. 1). Con-versely, the upper limit of available water lies at 86% WC in thebottom peat and 84% WC in the more decomposed, thin-layeredsurface peat (Fig. 1). In peat soils, a minimum air space of 10% soilvolume is considered critical for normal root development andplant growth (Päivänen, 1973). If the volume of air is lower thanthis, then poor aeration becomes a growth-limiting factor.

2.2. Chlorophyll fluorescence and soil water content measurements

Chlorophyll fluorescence was measured every 5–10 days fromdetached old (previous-year) needles starting June 1st and fromnew (current-year) ones starting June 30th until the end of August.New needles were technically too small and immature to measure

Fig. 1. Water retention at desorption of the 15-cm-thick bottom and 5-cm-thick surfacecylindrical soil samples. The water availability distinctions at corresponding units of pre

prior to June 30th. One new needle from the current-year leadershoot and one old needle from the previous-year leader shootper seedling were plucked approximately midway up the respec-tive shoots between 08:00 and 09:00, placed in a plastic bag, andstored in a small cooler to await darkening and measurementwithin 6 h. Mohammed and Noland (1997) recommended such astorage procedure. Needle detachment has been shown to haveno effect on the chlorophyll fluorescence of Scots pine (Otronenand Rosenlund, 2001; Percival and Sheriffs, 2002). Furthermore,our preliminary testing of the needle sampling procedure demon-strated that the leaf clips were too heavy for needles attached tothe young seedlings to bear. Altogether, chlorophyll fluorescencewas measured 13 times from old needles and 9 times from newones during the study.

After collection, the needles were dark adapted for half an hourin leaf clips equipped with a shutter plate inside a black,light-impenetrable bag at room temperature 20 �C. Thereafter,chlorophyll fluorescence parameters were measured using thenon-modulated Plant Efficiency Analyzer (Hansatech InstrumentsLtd., UK) with its probe set at 100% of maximum light (i.e., saturat-ing) intensity for 15 s. The probe’s six light-emitting diodes (LEDs)illuminate the leaf surface with red light having a peak wavelengthof 650 nm. The parameters derived from the induction kinetics ofchlorophyll fluorescence were as follows: Fo = minimal fluores-cence in the dark-adapted state; Fv = yield of variable fluorescencein the dark-adapted state (Fv = Fm � Fo); Fm = maximal fluorescencein the dark-adapted state; Fv/Fm = photochemical trapping effi-ciency in the dark-adapted state, i.e., the maximum potential quan-tum efficiency of PSII if all capable reaction centers were open. InSection 3, we will focus on interpreting the parameter Fv/Fm.

Immediately after fluorescence measurements, the volumetricwater content (WC, %) of each peat block was measured using a soilmoisture meter equipped with a sensor (Moisture Meter HH2 andThetaProbe ML2x, Delta-T Devices Ltd., UK). Default coefficient val-ues for organic soil were used (derived from Roth et al., 1992). Themanufacturer reports a loss of absolute accuracy up to 4% volumet-ric WC with this meter setting. The sensor bears four, 6-cm-longspikes, which are inserted into the soil. The top 6 cm of each peatblock was measured from the same four spots every time ofmeasurement to obtain the average moisture content. Towardsthe end as the blocks in the drought treatment severely dried, itwas necessary to drill the holes before WC measurement due toshrinking. The saturated soil in the wet treatment occasionally

Sphagnum peat components of the blocks. Each dot represents the mean ± SE of fivessure have been modified from Päivänen (1973).

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led to over-range moisture readings, which were interpreted as100% WC in the data.

2.3. Shoot characteristics

At the conclusion of the experiment, eight different shoot traitswere surveyed. Height, length of the current-year terminal leadershoot, diameter (D) at midsection of the current-year and previ-ous-year terminal leader shoots, and apical bud length were mea-sured from all seedlings. Diameter was determined using a pair ofvernier calipers. In addition, current-year needle length and freshmass (FM), and FM of the terminal bud group were measured froma sample of five seedlings per treatment. To determine needlelength, 20 new needles from the current-year terminal shoot ofeach sampled seedling were removed with tweezers and measuredwith a ruler, the mean value then calculated per seedling andthereafter by treatment. The needles were then weighed and thesum of their mass divided by 20 to attain needle FM. The terminalbud group was carefully excised with a scalpel from each of thesampled seedlings and scaled to determine FM.

2.4. Root characteristics and hydraulic conductance and conductivity

Four seedlings per treatment were selected for determining roothydraulic conductance (Kr). Kr (kg s�1 MPa�1) was measured with ahigh pressure flow meter (HPFM, Dynamax Inc., Houston, TX)(Tsuda and Tyree, 2000). For Kr measurements, the shoot of eachpotted seedling was excised about 1.5 cm above the root collarand the root connected to the HPFM. The root system was gradu-ally pressurized up to 0.5 MPa to obtain a pressure-flow relation-ship. Afterwards, roots were separated from the soil and theirlength, surface area, and projected area (i.e., volume) determinedby scanning (WinRhizo, Régent Instruments Inc., Québec, Canada).

Root hydraulic conductivity (Lp), i.e., the water flow rate perunit pressure scaled by root volume or surface area, was obtainedby dividing root conductance by root volume (kg s�1 MPa�1 cm�3)and root area (kg s�1 MPa�1 cm�2). For the sake of simplicity, themass unit in Kr and Lp has been converted to mg in applicable fig-ures and tables. Lastly, the roots were dried in an oven at 105 �Covernight and weighed to determine dry mass (DM). Altogethereight different root traits including conductance were analyzed.

2.5. Polyamine analysis

Free polyamine concentrations in current-year needles, termi-nal and lateral shoot tips of the topmost whorl (including needles,stems and buds), and fine roots were analyzed from five seedlingsper treatment at the end of the experiment (September 7th). Seed-ling root systems were rinsed off and placed in distilled water. Fineroots were then excised and dried with paper towel. All 45 sampleswere weighed (100–400 mg FM per sample) and stored in a freezer(�80 �C) until analysis. Polyamine samples were ground in liquidnitrogen and extracted with 5% (w/v) HClO4 after which free poly-amines from the crude extract were dansylated and separated withHPLC (Merck-Hitachi) (Fornalé et al., 1999; Sarjala and Kaunisto,1993). The putrescine, spermidine and spermine concentrationsare expressed as nmol g�1 FM of plant tissue.

2.6. Statistical analyses

The analysis of the chlorophyll fluorescence parameter Fv/Fm

and free polyamine concentrations were based on linear mixedmodels (procedure MIXED in SPSS 17, SPSS Inc., Chicago, IL, USA).The model was structured as follows:

Y ¼ mþ T þ N þ Dþ T � Dþ N � Dþ T � N þ T � D � N þ sþ e;

where Y is the response variable (Fv/Fm, free putrescine, spermidine,or spermine concentrations), m the constant, T the treatment, N theneedle age class (or sample type for polyamine testing), D the time(only for chlorophyll fluorescence testing), s the seedling and e isthe residual term.

Treatment (T), needle age class (or sample type for polyaminetesting) (N), and time (only for chlorophyll fluorescence testing)(D) were treated as fixed effects and seedling as a random effect.The models define a split-plot structure with needle age (or sampletype) as a split-plot treatment. A first-order autoregressive (AR1)covariance structure was assumed for the time correlationbetween the residuals of a needle age class of a seedling. TheBonferroni adjustment method was applied to the confidenceintervals and significance values to account for multiple compari-sons. Variances of the residuals of the fluorescence parameter(Fv/Fm) depend on the expected values and the fluorescenceparameter values were transformed using an arcus sine square roottransformation. In spite of the transformation, the variances of theresiduals were dependent on the predicted values, which wastaken into account by using regression weights w = 1/(predu *(1 � predu), where predu is a predicted value computed byunweighted analysis. Normality and homogeneity of the varianceof the residuals were checked graphically and the selection of thecovariance structure was based on Akaike’s information criteria.The fluorescence data on old needles for the period 1.–24.6 wasexcluded from the statistical analysis because otherwise the modelcould not have considered the effect of needle age correctly due tothe unequal number of measurements.

The distributions of all root and shoot traits were tested for nor-mality and equality of variances before running one-way ANOVA totest the significance of the treatment effect using the same statis-tical package mentioned above. The Bonferroni method was usedas the post hoc test for multiple comparisons.

The level of significance applied in all testing was 0.05.

3. Results

3.1. Soil water content and photochemical efficiency

The soil water conditions leading up to the onset of stress areshown in Fig. 2. Overall treatment had a significant effect on Fv/Fm (Table 1). Both drought and waterlogging stress significantly re-duced Fv/Fm relative to the control (p = 0.000 in both cases). Theimpact of treatment, however, depended on needle age (Table 1).The significantly lower Fv/Fm values of stressed seedlings were ob-served namely in old needles (mean ± SE as follows: DT0.835 ± 0.002; WT 0.836 ± 0.001; CT 0.844 ± 0.001; p = 0.000 inboth cases). Thus, overall Fv/Fm in new needles of stressed seed-lings did not markedly differ from that of the control (DT0.844 ± 0.001; WT 0.844 ± 0.001; CT 0.848 ± 0.001; p = 0.148 inboth cases). In addition, Fv/Fm was significantly higher in new asopposed to old needles for all treatments (p = 0.000 within DTand WT, p = 0.029 within CT). Yet both stressors seemingly wid-ened the gap in Fv/Fm between old and new needles compared tothe control.

Though no immediate impact was apparent, unfavorable soilwater conditions eventually caused a sustained decrease in Fv/Fm.Consequently, treatment and needle age also interacted signifi-cantly with time (Table 1). The first indication of stress in old nee-dles occurred in the wet treatment after the seedlings had spent2 months in saturated soil conditions (Figs. 2 and 3). From thatpoint onwards (August 5th) (WT vs. CT, p = 0.008), Fv/Fm declinedsteadily and significantly in the old needles of wet-stressed seed-lings in respect to the control (WT vs. CT: August 12th, p = 0.020;August 19th and 27th, p = 0.000). Although a clear decrease in Fv/

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Fig. 2. Volumetric water content of the peat blocks in the three different treatmentsover the course of the study. Each symbol corresponding to given date representsmean ± SE. These water content values were obtained using a soil moisture meter.The dotted lines represent the critical levels of water availability which weredefined in Fig. 1 for the studied peat substrate. The upper limit approximates withexcess water and the lower limit with water shortage (i.e., wilting point) in the peat.

Table 1Fixed effects of linear mixed model for Fv/Fm.

Source df F p

Time 8 17.873 0.000Treatment 2 11.254 0.000Needle age 1 50.656 0.000Time * treatment 16 9.564 0.000Time * needle age 8 26.841 0.000Treatment * needle age 2 2.881 0.056Time * treatment * needle age 16 3.761 0.000

Fig. 3. Fv/Fm of dark-adapted previous-year and current-year Scots pine needles inthe three different treatments over the course of the study. Each symbolcorresponding to a given date represents mean ± SE.

Table 2Results of the one-way ANOVA for testing the treatment effect on various shootcharacteristics.

Trait df F p

Seedling height 2 3.890 0.026Current-year terminal shoot length 2 6.213 0.004Current-year terminal shoot diameter (D) 2 48.314 0.000Previous-year terminal shoot diameter (D) 2 20.029 0.000Needle length 2 5.211 0.024Needle fresh mass (FM) 2 8.648 0.005Apical bud length 2 64.976 0.000Terminal bud group fresh mass (FM) 2 28.231 0.000

M. Pearson et al. / Forest Ecology and Management 307 (2013) 43–53 47

Fm in old needles of drought-stressed seedlings did not emerge un-til 2 weeks later (August 19th) (DT vs. CT, p = 0.001) when soil WChad dropped to 14% (Fig. 2), the decline was more drastic than inthe wet treatment. By experiment’s end, Fv/Fm in old needles ofthe drought treatment was significantly lower than in the controland wet treatments alike (August 27th: p = 0.000 in both cases)(Fig. 3). In the case of new needles, the only significant change ob-served in Fv/Fm befell the drought-stressed seedlings at the end ofthe experiment (August 27th) when the soil had dried still furtherto 11% WC (Figs. 2 and 3). Similarly to old needles, Fv/Fm of newneedles in the drought treatment was significantly lower than inthe other treatments (August 27th: DT vs. CT, p = 0.000; DT vs.WT, p = 0.002) (Fig. 3).

On a separate note, early on in the experiment Fv/Fm tended tofluctuate before finally stabilizing; in old needles (period 1.–15.6)this was likely due to planting stress, and in new needles (period30.6–16.7) a consequence of needle immaturity (Fig. 3). Further-more, based on the levels of photochemical efficiency measuredat the end of the study, none of the seedlings had perished in eitherof the stress treatments.

3.2. Shoot traits

For all eight shoot traits measured, the effect of treatment wassignificant (Table 2). Namely, the consequences of the two types ofstress—too little vs. too much soil water—were dissimilar. Relativeto the control, drought-stressed seedlings were significantly short-er in height and length of current-year terminal shoots, new nee-dles, and apical buds (Fig. 4a, b, e, and f). In addition, both

current and previous-year terminal shoots were significantly thin-ner (Fig. 4c and d) while new needles and terminal bud groupslighter in the drought treatment than in the control (Fig. 4g andh). Seedlings in the wet treatment, however, did not differ signifi-cantly from the control for any of these traits. On the contrary,wet-stressed seedlings had significantly thicker current and previ-ous-year terminal shoots (Fig. 4c and d), longer apical buds, as wellas heavier needles and terminal bud groups than seedlings sub-jected to drought (Fig. 4e, g and h).

3.3. Root traits

In regards to root traits, significant differences were observedbetween treatments for root volume, dry mass, and hydraulic con-ductance (mg s�1 MPa�1) (Table 3). Once again, drought-stressedseedlings differed significantly from the control as drought clearlylowered root volume, dry mass, and conductance rate (Fig. 5a, e

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Fig. 4. Shoot traits of Scots pine seedlings according to treatment. Traits presented in (a–e) were measured from all 75 seedlings, and those in (f–h) were determined from asample of 5 seedlings per treatment. Significant differences between treatments at the 0.05 level are indicated with different letters (X and Y) at the bottom of the bars,whereas insignificant ones with the same letter (X and X or Y and Y). XY at the bottom of a bar indicates that the treatment in question differs from neither of the other twosignificantly.

48 M. Pearson et al. / Forest Ecology and Management 307 (2013) 43–53

and f). Waterlogged soil, on the other hand, did not yet appear tonegatively impact seedling roots. For example, the root dry massof wet-stressed seedlings was actually greatest of the three treat-ments on average (Fig. 5e), while also differing significantly fromthat of drought-stressed ones. Although the trends for root areawere very similar to those for volume (Fig. 5b), the treatment effectwas shy of significance (Table 3).

While treatment markedly influenced the rate of root hydraulicconductance (Kr), no such effect was observed when scaled byeither root volume (Lp, mg s�1 MPa�1 cm�3) or area (Lp, mg s�1

MPa�1 cm�2) (Table 3, Fig. 5g and h). Furthermore, neither the

Table 3Results of the one-way ANOVA for testing the treatment effect on various rootcharacteristics.

Trait df F p

Volume of roots 2 5.648 0.026Surface area of roots 2 3.974 0.058Length of fine roots (D < 1 mm) 2 2.618 0.127Length of roots 2 2.795 0.114Dry mass (DM) of roots 2 14.090 0.002Root hydraulic conductance, Kr (mg s�1 MPa�1) 2 5.062 0.038Root hydraulic conductivity, Lp (root volume-specific) 2 0.131 0.879Root hydraulic conductivity, Lp (root area-specific) 2 0.168 0.849

length of fine roots nor that of all roots combined was significantlyaffected by drought or waterlogging stress (Table 3, Fig. 5c and d).

3.4. Free polyamine concentrations in new needles, fine roots, andshoot tips

The impact of treatment on the free putrescine level was con-tingent on sample type (Table 4). Drought significantly increasedfree putrescine concentrations in new needles relative to not onlythe control (p = 0.004), but also the wet treatment (p = 0.003)(Fig. 6a). For fine roots and shoot tips, however, the differences be-tween treatments were not significant (roots: CT vs. WT, p = 0.145;CT vs. DT, p = 1.000; WT vs. DT, p = 0.077; shoots: CT vs. WT,p = 0.983; CT vs. DT, p = 1.000; WT vs. DT, p = 1.000) (Fig. 6b andc). Given the 63% higher concentration in roots of wet-stressedseedlings in respect to the control (Fig. 6b), it would nonethelessseem that waterlogged soil had at least a mild impact. The rootconcentration in the wet treatment was also 80% higher than forthe drought treatment.

Notably, the relationships between plant parts, i.e., sampletypes likewise varied according to treatment. Within the control,belowground organs differed significantly from aboveground parts(new needles and shoot tips > fine roots; p = 0.000 in both cases);in comparison, wet stress leveled off between part differences in

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Fig. 5. Root traits of Scots pine seedlings according to treatment. All root traits were determined from a sample of four seedlings per treatment. Significant differencesbetween treatments at the 0.05 level are indicated with different letters (X and Y) at the bottom of the bars, whereas insignificant ones with the same letter (X and X or Y andY). XY at the bottom of a bar indicates that the treatment in question differs from neither of the other two significantly.

Table 4Fixed effects of linear mixed models for free polyamines.

Source df F p

Free putrescineTreatment 2 0.965 0.409Sample type 2 46.641 0.000Treatment * sample type 4 7.187 0.001

Free spermidineTreatment 2 0.943 0.399Sample type 2 28.269 0.000Treatment * sample type 4 2.062 0.106

Free spermineTreatment 2 5.565 0.019Sample type 2 54.067 0.000Treatment * sample type 4 0.137 0.967

M. Pearson et al. / Forest Ecology and Management 307 (2013) 43–53 49

free putrescine such that none were significant (new needles = fineroots, p = 0.101; shoot tips = fine roots, p = 1.000), whereas droughtstress emphasized these differences such that all were significant(new needles > shoot tips and fine roots, shoot tips > fine roots;p = 0.000 in all cases) (Fig. 6a and c).

The free spermidine concentration did not markedly differ be-tween treatments, nor was treatment found to interact signifi-cantly with sample type (Table 4, Fig. 6d and f). Nevertheless, thedrought treatment did elevate spermidine levels somewhat

(though insignificantly) in new needles compared to the control(Fig. 6d).

Treatment was found to dictate the concentration of freespermine (Table 4). Independent of sample type, the concentrationof free spermine was significantly greater in the drought-stressedthan the control (p = 0.030) and wet-stressed (p = 0.050) seedlings(Fig. 6g–i). The wet treatment had no effect with respect to thecontrol (p = 1.000) (Fig. 6g–i).

4. Discussion

4.1. Impacts of water stress on Scots pine seedling performance

In examining the growth responses of Scots pine seedlings todrought and waterlogging, it is first necessary to consider its shootand root phenology (Iivonen et al., 2001). In our study, the approx-imate period for shoot elongation was late May until mid-July, andfor needle elongation mid-June to mid-August. Hence, growth wasstill in progress especially that of new needles, when soil water be-came a limiting factor which then climaxed in shorter current-yearneedles and shoots in the drought treatment. If we also considerthat a soil WC of about 38% in the Sphagnum (bottom) peat is thelimit when the water shortage presumably starts to restrict plantgrowth (�100 kPa, Fig. 1), then this limit was already reached bymid-July, almost 2 months before the experiment ended (Fig. 2).

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Fig. 6. Free polyamine concentrations (mean ± SE) in Scots pine seedlings according to treatment and sample type. Sample size five seedlings per treatment.

50 M. Pearson et al. / Forest Ecology and Management 307 (2013) 43–53

Thus, during their ‘‘window’’ of growth, there was simply not en-ough water available for roots to grow and this was reflected as re-duced root volume and dry mass in drought-stressed seedlings. Indrying soil, Kaufmann (1968) observed that a soil water potentialof �0.6 to �0.7 MPa (�600 to �700 kPa) encumbered root growthin loblolly and Scots pine seedlings to a rate of only 25% of that atfield capacity. Decreased shoot and root growth is a commonly ob-served response to drought amongst pine seedlings (Kaufmann,1968; Otronen and Rosenlund, 2001; Pearson et al., in press; Rikalaand Puttonen, 1988; Sword Sayer et al., 2005) and our study alsoconfirms this. Furthermore, the shorter apical buds and consider-ably lighter terminal bud groups indicate inadequate bud forma-tion in drought-stressed seedlings, which would have a carry-over effect into the next growing season. Thus, growth losses aresubstantial in Scots pine seedlings due to drought.

Although the Scots pine seedlings in the wet treatment spent3 months in waterlogged soil from start to finish, neither shootnor root growth was affected. According to Päivänen (1973), anair content lower than 10% of peat soil volume would hinder plantgrowth. Here, the peat WC in the wet treatment ranged between95% and 100% for most of the experiment. Though this reportedWC range obviously involves some error (refer to Section 2.2) sincepeat porosity was a maximum of 94–96%, the volume of air presenthad to be less than the minimum ‘‘guideline’’ value of 10% in anycase (upper limit of available water in the peat 84–86%, Fig. 1).Such nonresponsiveness to excess moisture has also been demon-strated earlier with Scots pine (Armstrong and Read, 1972; Mukas-sabi et al., 2012; Otronen and Rosenlund, 2001; Zaerr, 1983) andlodgepole pine (Armstrong and Read, 1972; Boggie and Miller,1976; Wolken et al., 2011). Several theories have been proposedto explain the tolerance of Scots pine to flooding. Armstrong andRead (1972) suggested an internal oxygen diffusion pathway fromshoot to root in anaerobic soil conditions. When placed in anaero-

bic soil, substantial oxygen diffusion from the roots of Scots pineseedlings was observed, which could be interpreted as successfuladaptation to wet soil. Coutts and Philipson (1978) also attributedthe growth of lodgepole pine roots into soil devoid of oxygen to aninternal oxygen transport mechanism. Another potential mecha-nism is the formation of hypertrophied lenticels on submergedstems and roots, which has been noted in many pine species (Koz-lowski, 1997; Topa and McLeod, 1986a,b) including Scots pine(Aronen and Häggman, 1994; Hahn et al., 1920). The developmentof aerenchymatic tissue in the stems and roots of several pine spe-cies has also been identified as an effective strategy in dealing withwaterlogged soil (Philipson and Coutts, 1980; Topa and McLeod,1986b; Wolken et al., 2011). Each of the aforementioned adapta-tion strategies either alone or in combination would serve to facil-itate gas exchange (and possible dispersion of ethylene) andaeration of submerged roots at least in the short term.

While Scots pine seedlings in our study exhibited resilience towaterlogged soil, we have reason to suspect that some oxygenwas still available in the topmost part of the saturated peat blocks.Since the water level was maintained so as to submerge the bot-tom half of the seedling pot, 6–7 cm of the peat block remainedabove the water level on all four sides. Even though capillarymovement of water in the peat kept it saturated as demonstratedby volumetric moisture measurements, it is possible that the out-ermost portions of the block (above the water level) were subjectto some degree of evaporative force, hence being slightly drierand thus better oxygenated than the inner core of the block. Onthe other hand, the thick PVC frame enveloping each block wouldhave minimized this evaporative loss. However, Mukassabi et al.(2012) pointed out that Scots pine seedling roots were consider-ably shorter and lower in mass namely when the water tablewas 0–4 cm below the soil surface rather than 6.6 cm. In the lattercase, seedling roots resembled those in the control treatment,

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M. Pearson et al. / Forest Ecology and Management 307 (2013) 43–53 51

which corresponds well with our findings. Although we attemptedto emulate excess moisture stress commonly encountered in fieldconditions, one drawback of our experimental design is that it doesnot consider the input of water from above through precipitation(and runoff) as we never watered the seedlings from the top.

When considering the metabolic response of Scots pine seed-lings to water-associated stress, the impact of drought was alsomore pronounced than the wet treatment. Drought increased freeputrescine levels in new needles as well as those of spermine innew needles, shoot tips, and fine roots relative to the control treat-ment, whereas the wet treatment did not significantly differ fromthe control for any of the free polyamines or sample types ana-lyzed. These polyamine changes in drought-stricken needles oftwo-year-old Scots pine seedlings were in line with our earlierstudy (Pearson et al., 2013) with year-old seedlings, in which how-ever, putrescine concentrations collapsed in the new needles at theend of the experiment under extreme drought when soil water wasno longer available (i.e., well below the permanent wilting point ofthe peat in question). Contrary to the observations of Pearson et al.(2013), spermidine concentrations were not affected by thedrought treatment in this study. However, in our previous studythe higher spermidine level in the roots of sufficiently wateredone-year-old seedlings was attributed to the significantly highernumber of mycorrhizal root tips, which are known to increasespermidine concentrations in Scots pine seedlings (Niemi et al.,2006; Sarjala et al., 2010). In the present study, mycorrhizal roottips were not counted, but no significant effect of drought stresswas found on the length of fine roots at the end of the experiment,which may partly explain the lack of an effect on the spermidinelevel in the roots. Furthermore, seedlings were older, thus perhapsmore resistant, and the drought inflicted was not quite as severe asin Pearson et al. (2013) as evidenced by the higher photochemicalefficiency of drought-stressed old and new needles in the presentcompared to the previous study. The role of both spermidine andspermine has been suggested to be connected to regulation ofsenescence (Moschou et al., 2012) and furthermore, spermine hasbeen shown to protect leaves from decay of photosystem com-plexes (Serafini-Fracassini et al., 2010). In this study, spermine con-centrations were increased due to drought in all seedling parts.Interestingly, drought stress also amplified the difference in poly-amine concentrations between the above and belowground plantparts mostly by elevating concentrations in the needles. This sug-gests that the aboveground parts of the seedlings were preferredto maintain functionality while roots were more susceptible tosenescence under extreme drought stress.

Based on our results here and in our previous study (Pearsonet al., 2013), water stress in Scots pine seedlings has to be severebefore needle photochemical efficiency is impacted. Similarly,Otronen and Rosenlund (2001) found no impact on Fv/Fm ratiosof current-year needles in Scots pine seedlings grown at five differ-ent soil moisture levels ranging from 10% to 80%. They did not,however, monitor Fv/Fm of previous-year needles. On the same line,Binder et al. (1996) observed a drop in Fv/Fm only after severedrought in jack pine (Pinus banksiana Lamb.), while no indicationof water stress in the Fv/Fm ratio was evident in Masson pine (Pinusmassoniana L.) seedlings subjected to wet, medium, and dry soilmoisture treatments (Fang-yuan and Guy, 2004). If we considerthe availability of water for plant use in the given peat substratein our study, the degree of soil drying necessary to reduce Fv/Fm

was just at or around the permanent wilting point, 10–12% WC(14% peat WC when it changed in old needles, and 11% when itdid so in new needles) (Figs. 2 and 3). This may account for therather drastic nature of the collapse particularly in old needles ofdrought-stressed seedlings.

In any case, our results show that the integrity of PSII in Scotspine is not easily compromised, and this would likely aid

post-stress recovery in the event that soil water availability wouldonce again improve. On the whole, wet-stressed seedlings did notseem particularly distressed physiologically even though Fv/Fm ofold needles indicated that something was wrong. After having togrow for 3 months in waterlogged peat soil during the growingseason, i.e., when most vulnerable, the undaunted root and shootgrowth combined with the almost cosmetic effect on Fv/Fm inwet-stressed seedlings indicated remarkable tolerance to saturatedsoil conditions. Such resilience has also been noted by Mukassabiet al. (2012), who reported high Scots pine seedling survival rateseven after 25 dormancy-free months in waterlogged soil. WhileScots pine seedlings do not appear to be vulnerable to waterlog-ging stress in the short term, their response in the long term re-mains to be studied. Nevertheless, as mentioned earlier, we mustbe conservative in the interpretation of our results since it maybe that only partial flooding was achieved in our experiment.

In congruence with our previous study (Pearson et al., 2013),previous-year needles gave way first, i.e., incurred reduced photo-chemical efficiency in both stress treatments. This observationconforms with the known tactic of shifting growth resourceswithin Scots pine, e.g., nutrient retranslocation from old needlesto developing new needles and elongating shoots (Fisher andHöll, 1991; Helmisaari, 1992). Under water stress, current-yearneedles were in effect prioritized, which is logical when consider-ing that most net photosynthesis is due to current-year needles inScots pine in the latter half of the growing season (Ericsson, 1979;Gezelius and Hallén, 1980; Iivonen et al., 2001; Vapaavuori et al.,1995).

In our study, only drought lowered Kr of two-year-old Scotspine seedlings (Fig. 5f). However, when Kr was scaled by root vol-ume or surface area, no significant differences in root conductivity(Lp) were observed between treatments (Table 3, Fig. 5g and h).Decreased Kr was accompanied by corresponding reductions inroot dry mass, surface area, and volume in drought-stressed seed-lings. Thus, by the time root conductance and conductivity weremeasured, their roots were obviously under considerable stressand some of them had likely already become damaged conse-quently losing their resistance mechanism. A loss in resistancedue to root tissue damage and cell death would entail increasedmovement of water into roots and thus reflect as increased Lp. AsKramer and Boyer (1995) noted, the movement of water into rootsis decreased by stress treatments, but conversely increased by kill-ing the roots. Similar conclusions as ours have been previouslyreached by at least Apostol and Zwiazek (2003) for jack pine andElse et al. (1995) for tomato (Lycopersicon esculentum Mill. cv AilsaCraig) plants although with different types of stress.

Notably, Kr and Lp of two-year-old Scots pine seedlings were notaffected by waterlogged soil here. In the study of Apostol andZwiazek’s (2003) mentioned previously, purely hypoxic conditionshowever reduced root dry mass and both Kr and Lp in half-year-oldjack pine. Our findings agree with Reece and Riha (1991) but con-trast with those of Islam et al. (2003, 2004) for tamarack, a typicaltree species on boreal peatlands in North America. Seedling age(and thus degree of tolerance) may account for some of this incon-gruence, as Reece and Riha (1991) used two-year-old while Islamet al. (2003, 2004) 6 and 18-month-old seedlings. Even withinthe latter mentioned studies Kr ranged from 5–8 mg s�1 MPa�1 inthe flooded 18-month-old tamarack seedlings but only from 2.5to 4.5 in the half-year-old ones. Unlike in our study, these seedlingswere submerged to the root collar level.

4.2. Implications for soil management and forest regeneration on peatsoils

With this experimental design, we attempted to mimic thestress encountered by Scots pine seedlings in the field in different

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52 M. Pearson et al. / Forest Ecology and Management 307 (2013) 43–53

mechanically prepared microsites on peat soil (see e.g., Pearsonet al., 2011)—desiccation of mounds vs. waterlogging of scalps.Neither excess drying of prepared heaps of peat, i.e., mounds(Saarinen, 2005) nor flooding of flat, bare peat microsites fromwhich the humus layer has been removed, i.e., scalps is uncommon(Pearson et al., 2011) in drained peatland forest regeneration areasin the present climate of the boreal zone. Hence, in the future anincrease in the incidence of extreme weather events may aggravatethese conditions further.

To reduce the negative impacts of drought on peat-grown Scotspine seedlings demonstrated here, we suggest applying a mound-ing technique in peatland forest regeneration areas which (1)creates low peat mounds less exposed to drying, and (2) does notcut off capillary movement of water from the peatland water table(i.e., the bucketful of peat is turned upside down back onto the spotfrom which it is excavated rather than being dropped onto a veg-etated, intact surface or harvesting residues). Inverted mounds,as opposed to ditch mounds, would at least delay the ill effectsof water shortage on seedling roots. This would be especiallybeneficial for newly planted tree seedlings, which tend to be mostvulnerable to drought before becoming sufficiently established intheir planting spots.

In regards to the impacts of waterlogged peat soil on seedlings,our experiment provided little evidence of suffering. Based on ourresults, one growing season in exceptionally wet soil conditionswould seem to have little impact on Scots pine seedlings. However,we must again emphasize two points: (1) this was a short-term,semi-controlled experiment, and (2) partial flooding may in partexplain the absence of negative effects. As Pearson et al. (2011)found, the combination of a high water table level and copiousrainfall in back to back growing seasons can lead to disastrousregeneration results for Scots pine outplants in scalped micrositeson forestry-drained peatland. However, in that study, the profile ofscalps was not consistently flat, rather depressions were made intothe peat which then collected runoff, hence compounding the dif-ficult situation further. In any case, we must stress the importanceof carrying out both short and long-term experiments in field con-ditions in order to distinguish the ‘‘outer limits’’ of waterloggingtolerance of Scots pine for the benefit of practitioners involved inpeatland forest regeneration.

5. Conclusions

In our study, the effects of desiccated rather than waterloggedpeat soil on two-year-old Scots pine seedlings were more apparentin the short term. Over one growing season, drought distinctly re-duced seedling vitality and root and shoot growth whilst wet stresshad little discernible impact. Despite the dissimilar consequencesof drought vs. waterlogging stress, it is important to note that noneof the seedlings had died by the end of the study in either stresstreatment. Hence, Scots pine seedlings demonstrated high toler-ance to adverse soil water conditions. Apparently, waterloggingdoes not modify seedling growth or vitality immediately, butrather in the longer term. Our findings thus suggest the existenceof a ‘‘window for recovery’’ in wet-stressed Scots pine seedlings;should a wet growing season be followed by a relatively normalone, then the consequences for vitality and growth are likely min-imal. The lack of fatalities and maintenance of rather high photo-chemical efficiency despite severe drought stress would seem toindicate a potential for seedling recovery if water availability inthe peat substrate improved. However, growth losses had alreadymaterialized. In the future, it will be necessary to examine the vul-nerability of Scots pine seedlings to long-term drought and water-logging in peat as well as recovery in both field and greenhouseenvironments.

Acknowledgements

This study was made possible with the financial support pro-vided by the Finnish Cultural Foundation, Research Foundation ofthe University of Helsinki, Maj and Tor Nessling Foundation, GSFor-est – Graduate School in Forest Sciences, and Niemi Foundation.We also thank Eeva Pihlajaviita for performing the polyamine anal-ysis and Jaakko Heinonen for valuable statistical advice.

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