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EffectsoftimingofsoilfrostthawingonScotspine

ARTICLEinTREEPHYSIOLOGY·SEPTEMBER2005

ImpactFactor:3.66·DOI:10.1093/treephys/25.8.1053·Source:PubMed

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TapaniRepo

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TimoDomisch

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HannuMannerkoski

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LeenaFinér

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Summary Effects of the timing of soil thawing in the springon Scots pine (Pinus sylvestris L.) were studied under con-trolled laboratory conditions. Sixteen 6-year-old saplings werelifted from the field, replanted in containers and placed in fourtreatments in controlled environment (CE) chambers with fourreplicate saplings per chamber. The saplings were held in theCE chambers during one simulated winter and one simulatedgrowing season. The soil was frozen to –2 °C during a secondsimulated winter in the CE chambers, and the soil thawingtreatments began at the end of the second simulated winter. Soilthawing began at various times before (no delay in thawing)and after (delay in thawing) chamber air conditions werechanged from simulated winter to simulated summer. Delayedsoil thawing subjected saplings to stress, with the severity ofstress depending on the length of the delay in thawing. If therewas no delay or only a short delay in soil thawing, stress wasminor and reversible. A 2-week delay in soil thawing led todeath of the saplings. Stress was apparent as decreases in thevariable to maximal chlorophyll fluorescence ratio (Fv/Fm),chlorophyll a/b ratio and needle water potential. In needles ofstressed saplings, apoplastic electrical resistance first de-creased and then increased and there were anomalies in theelectrical impedance spectra of the stems. Stress from the soilthawing treatments affected both root and shoot growth.

Keywords: carbohydrate, chlorophyll, chlorophyll fluores-cence, electrical impedance, growth, minirhizotron, phenol-ogy, Pinus sylvestris, root, soil freezing, water potential, waterstress.

Introduction

Soil frost has a multitude of consequences for both soil andvegetation (Archibold 1995, Sutinen et al. 1998, Solantie2000, Groffman et al. 2001). It may affect root and microbialmortality, soil mineralization, retention or leaching of nutri-ents and soil–atmosphere trace gas fluxes (Groffman et al.2001). In the boreal forests of Finland, soil frost limits treegrowth, partly by shortening the growing season (Solantie2003).

In boreal areas, the annual duration of soil frost varies from

zero to 300 days, although in certain areas, permafrost maypersist throughout the year beneath the root layer. The depth ofsoil frost depends on air temperature, snow depth, vegetation,soil texture and water content (Sakai 1970, Fahey and Lang1975, Solantie 2000, Venäläinen et al. 2001a). In Finland(60–70° N), forest soil freezes to a maximum depth of 1 to 2 m,depending on latitude and elevation, with a mean annual maxi-mum depth of 15 to 150 cm (Huttunen and Soveri 1993). De-pending on location, soil thawing starts at different times andwith different delays following the occurrence of air tempera-tures high enough for shoot growth (Solantie 2003).

In spring, high daytime air temperatures favor the initiationof CO2 assimilation and growth, but cool or frozen soil inhibitsphotosynthesis by restricting or preventing the uptake of waterand nutrients (Bergh et al. 1998, Bergh and Linder 1999, Jarvisand Linder 2000, Strand et al. 2002). Under such conditions, ifsaplings are not covered by snow, evapotranspiration is high. Ifwater uptake by roots cannot take place because of soil frost,water stress occurs, leading to embolization of vascular tissueand dessication injury to leaves (Tranquillini 1982, Larcher1985, Larsen 1993, Kramer and Boyer 1995, Boyce andLucero 1999). Such effects are common in Scots pine, espe-cially in the northern part of Finland, where brown leaders canbe found almost every year (Jalkanen 1993). If snow depth issubstantial, soil temperature may stay above 0 °C over thewinter and liquid water may be available to the roots early inthe spring, thus preventing the development of severe waterdeficit (Sutinen et al. 1998, Solantie 2000, Suni et al. 2003).

We attempted to follow the effects of water stress in Scotspine saplings in response to delayed soil thawing. Experimentswere carried out in controlled laboratory conditions wherethawing of frozen soil was initiated at different times relativeto an increase in air temperature from simulated winter to sim-ulated growing season conditions. Physiological and pheno-logical responses and growth of roots and shoots of saplingswere monitored.

Materials and methods

The study was conducted with 16 six-year-old Scots pine(Pinus sylvestris L.) saplings that were lifted from a plantation

Tree Physiology 25, 1053–1062© 2005 Heron Publishing—Victoria, Canada

Effects of timing of soil frost thawing on Scots pine

TAPANI REPO,1,2 TUOMO KALLIOKOSKI,1 TIMO DOMISCH,1 TARJA LEHTO,3 HANNUMANNERKOSKI,3 SIRKKA SUTINEN1 and LEENA FINÉR1

1 The Finnish Forest Research Institute, Joensuu Research Centre, P.O. Box 68, FIN-80101 Joensuu, Finland2 Corresponding author (tapani.repo@metla.fi)3 University of Joensuu, Faculty of Forestry, 80101 Joensuu, Finland

Received March 18, 2004; accepted November 20, 2004; published online June 1, 2005

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in eastern Finland (62°36′ N, 29°43′ E, 91 m a.s.l.) in fall 2000.The stand was regenerated with 2-year-old container-grownseedlings of local origin in 1997. According to the Finnishclassification system, the site is a poor Vaccinium type(Cajander 1949). The soil is fine-textured sand with 81% ofthe particles between 0.06 and 0.63 mm in size (pH and ele-ment concentrations of the soil are shown in Table 1). Meanheight and stem diameter (± SD) (15 cm above root collar) ofthe selected saplings were 92 ± 4.4 and 2.4 ± 0.2 cm, respec-tively.

Sixteen saplings were transplanted to a 0.46 m3 cylindricalcontainers (height 1.3 m, diameter 0.7 m) filled with fine-tex-tured sand to a height of 100 cm. The saplings were plantedabove the sand layer in mineral soil from the plantation wherethe saplings originated. A 10-cm-thick organic layer from thesame site was then placed on top of the mineral soil layer. Fourpotted saplings were transferred to each of four growth cham-bers (Model RTR48, Conviron, Winnipeg, MB, Canada),where air and soil temperatures were controlled independently(Finér et al. 2001). Soil water content was continuously moni-tored in the organic layer (ThetaProbe, ML2x, Delta-T De-vices, Cambridge, U.K.) and at a depth of 12 cm in the mineralsoil (CS615, Campbell Scientific, Shepshed, U.K.). Soil tem-perature was monitored in the organic layer and at differentdepths in the mineral soil. Each pot was equipped with twoglycol circulation coils, one at the bottom and another abovethe organic layer, to control soil temperature (see Finér et al.2001 for details). A sheet of insulating material was placedover the upper coil. At the final harvest, soil layers in the rootcontainers were sampled and tested for pH and analyzed formineral nutrient content.

The study comprised two 14-week simulated growth peri-ods (GS1 and GS2) and two 8-week simulated winter periods(SW1 and SW2) (Table 2). There was a 3-week short-dayphase (SD1) at the end of GS1. The soil was frozen duringSW2 (Figure 1). The effect of the timing of soil thawing wastested during GS2 (Table 3, Figure 1). Because of the thermalcapacity of the soil, the rise in soil temperature followed the in-crease in air temperature only after a delay. In all treatments,air temperature was changed gradually over 6 days between

each SW and GS. Sapling responses were monitored until theend of GS2. The needles that developed during GS1 aretermed GS1 needles.

Saplings were irrigated twice a week during each GS andevery 2 weeks during each SW, except when the soil wasfrozen. The composition of the irrigation solution for each GScorresponded to the annual precipitation in southern Finland.The temperature sum (degree days) and chilling unit (CU) ac-cumulation were 1230 d.d. and 50 CU for the each GS and SW,respectively (Sarvas 1974, Hänninen 1990). Calculation of thetemperature sum was based on the daily mean air temperature,with a threshold of 5 °C. Calculation of CU was based on thefunction determined for the bud dormancy release of borealtree species. The CU accumulation followed a piece-wise lin-ear function between daily mean temperatures of –3.4 and10.4 °C. Below and above these temperatures the accumula-tion rate is 0 CU day –1, with a maximum rate of 1 CU day –1 at3.5 °C (Sarvas 1974).

Physiology

Thirty GS1 needles from the second and third whorls from thetop of each sapling were sampled eight times for chlorophyllfluorescence. Dark-adapted (20 min) needles (two replicates)were measured at room temperature with a portable chloro-phyll fluorometer (MINI-PAM, Heinz Walz Gmbh, Effeltrich,Germany). The ratio of variable to maximal chlorophyll fluo-rescence (Fv/Fm) was taken as a measure of the potential pho-tochemical efficiency of PSII. The first measurements tookplace during SD1 and the later measurements were made dur-ing SW2 and GS2.

Chlorophyll a and b concentrations of the GS1 needles weredetermined seven times during SW2 and GS2 by the methoddescribed by Porra et al. (1989). Needles were sampled fromthe second and third whorls from the top of each sapling. Oneach occasion, 16 needles were sampled, frozen in liquid ni-trogen and stored at –80 °C. Frozen needles were ground in acold mortar and chlorophyll a and b extracted in cool 80% ace-tone and determined spectrophotometrically at 646.6, 663.6and 750 nm.

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Table 1. The pH and mineral nutrient content of the organic layer and the mineral soil at the site from which the experimental trees were obtained,and from the containers in which the experimental trees were grown. Mineral soil nutrient concentration was determined by the analysis of ammo-nium-acetate extracts.

Soil layer Soil pH N (%) Nutrient concentration (mg kgDM1− )

Ca Mg K P Fe Mn

FieldOrganic 3.7 0.71 1380 212 414 121 10 178Mineral (0–10 cm) 4.8 0.04 23 3.0 9.7 7.0 130 16Mineral (10–20 cm) 4.8 0.04 16 2.3 9.7 11 107 19

Container rooting mediumContainer sand (start) 5.8 0.01 94 14 12 3.5 15 0.4Organic (end) 4.0 0.85 1578 251 462 151 6.4 279Mineral (end, 0–10 cm) 4.9 – 194 29 59 20 240 103

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Eight pairs of GS1 needles were sampled from the secondand third whorls from the top of each sapling on each of nineoccasions during GS1, SW2 and GS2 for electrical impedancespectroscopy (EIS) and water potential measurements. Oneneedle of each pair was immediately taken for EIS and the wa-ter potential of the other needle was measured with a pressurechamber (Scholander et al. 1964). The EIS of needles was per-formed as described in Repo et al. (1994). A 10-mm sectionwas cut from the middle of the needle, and the cut surfaces ofthe needle section were placed in the measuring cell betweenthe electrode pastes (Signagel, Parker Laboratories, Fairfield,NJ). The pastes were connected by Ag/AgCl electrodes (RC1,WPI, Sarasota, FL) to the circuit analyzer (HP 4284A, Agil-ent, Palo Alto, CA) and the impedance spectrum (real andimaginary parts) measured at 46 frequencies between 20 and1 MHz. The extracellular resistance of the needles, which wascalculated according to the estimated parameters of the singleDCE model, corresponds to the value of the real part of the im-pedance spectrum when the frequency approaches zero (Repoet al. 1994). The extracellular resistance was normalized withrespect to the cross-sectional area and length of the sample toget a specific value (Ωm). The electrical impedance spectra(IS) of the stems were measured 6 weeks after the start ofthe GS2 period with two silver needle electrodes (diameter0.5 mm), 15 mm apart, that were pushed to a depth of 2 mminto the stem 15 cm above the root collar.

Soluble sugar and starch concentrations of GS1 needleswere determined five times during the study, following Han-sen and Møller (1975). On each occasion, 16 needles weresampled from the second and third whorls from the top of eachsapling, dried to a constant mass (40 °C) and ground to a pow-der. Soluble sugars were extracted from the samples in 80%aqueous ethanol. Total soluble sugars were determined colori-metrically at 630 nm by the anthrone method. Starch wasextracted from the residue with 35% perchloric acid and deter-mined colorimetrically at 625 nm by the anthrone method.Starch in 30% perchloric acid was used as a standard.

Shoot and root growth and phenology

Length of the main shoot, lengths of the branches and needlesin the two uppermost whorls and the diameter of the stem

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EFFECTS OF SOIL FROST THAWING ON SCOTS PINE 1055

Table 2. Chamber conditions during the experiment. Abbreviations:SW1, SW2, GS1 and GS2 refer to the first and second simulated win-ter and growing periods, respectively; LD1 = long-day phase; SD1 =short-day phase; RH = relative humidity; and PAR = photosyn-thetically active radiation.

Variable SW1 GS1 SW2 GS2

LD1 SD1

Air temperature 4 20/15 20/15 4 20/15(day/night) (°C)

RH (day/night) (%) 90/90 70/80 70/80 90/90 70/80

PAR (max.) 200 400 200 200 400(µmol m–2 s–1)

Photoperiod 6/18 18/6 6/18 6/18 18/6(day/night) (h)

Soil temperature 4 15 15 –2… –4 15(min.) (°C)

Duration (weeks) 11 11 3 8 11

Figure 1. Daily mean (n = 4) air and soil temperatures (A) and volu-metric water content of the organic layer (B) in the experiment withdifferent times for the onset of soil thawing in the treatments T1, T2,T3 and T4 (see arrows in Figure A). Vertical lines indicate the cessa-tion and start of the growing periods GS1 and GS2, respectively, withthe simulated winter period SW2 in between. Time refers to days fromthe beginning of the experiment (sampling at 20 min intervals).

Table 3. Timing of the soil thawing treatments in relation to thechange in air temperature. The increase in air temperature, photo-period and photon flux occurred at the same time in all of the treat-ments.

Treatment Description

T1 Soil thawing preceeded the rise in airtemperature by 14 days

T2 Soil thawing and increase in air temperaturewere simultaneous

T3 Soil thawing followed the increase in airtemperature by 14 days

T4 Soil thawing followed the increase in airtemperature by 24 days

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(15 cm above the root collar) were measured at 1-week inter-vals. The times of growth initiation and cessation were deter-mined as the times when 10 and 90%, respectively, of the finalextension had been reached. Shoot biomass was determined atthe beginning (four extra saplings), and shoot and root bio-mass at the end of the experiment on a dry mass basis (40 °C).Shoot biomass was determined for the live and dead parts sep-arately. To determine root biomass, two sectors of the soil(385 cm2 per sector) were sampled to a depth of 30 cm on op-posite sides of the stem. The roots were separated from thesoil, the dry mass of the sample determined and extrapolatedfor the whole container.

Root growth was monitored with a minirhizotron imagingsystem (Bartz BTC-100X Camera System, Bartz Technology,Santa Barbara, CA). The acrylic minirhizotron tube (outer andinner diameter 60 and 50 mm, respectively) was located hori-zontally at a depth of 5 cm in the mineral soil of each root con-tainer (Finér et al. 2001). Digital images of the root systemwere taken in an upward direction throughout the whole exten-sion of the tube in a total of 56 frames. Imaging took placeonce during GS1 and once during SW2. Thereafter, imagingwas repeated at 1-week intervals during GS2. The imageswere analyzed using RootView software (Aphalo and Simonic1999). Live root tips were counted, and the total lengths ofshort roots, which were mostly mycorrhizal, and long rootswere measured. The sum of the root tips and the total length ofthe short and long roots in all frames were calculated for eachroot container at each imaging time. The number of root tipsand the lengths of the short and long roots at the end of GS1were taken as the reference when the cumulative number wascalculated for each container during SW2 and GS2.

Statistical analysis

Treatment effects on chlorophyll fluorescence (Fv /Fm), chlo-rophyll a/b ratio, extracellular resistance, water potential,starch and soluble sugar concentration of needles, as well asroot-tip formation and root elongation, were analyzed with alinear MIXED model, with the assumptions necessary for thesplit-plot analysis of variance. The model was y = (µ + treat-ment + sampling + treatment(sampling) + replicate(treat-ment)), where µ is a constant. The “treatment” (i.e., differenttimes of soil thawing) and “sampling” (i.e., sampling time)were considered to be fixed factors and the “replicate” (i.e.,sapling) a random factor. The analysis was made for thosesampling times where the data for all treatments were avail-able. The significance of the difference between the treatmentsat different sampling times was tested by contrasts usingBonferroni corrected significance levels, i.e., the P value ofeach contrast was multiplied by the number of comparisons(number of contrasts × number sampling times). The differ-ence between the treatments at the start and cessation ofgrowth and in the chemical composition and pH of soil in theroot containers at the end of the study was analyzed by theMann-Whitney U-test. Differences in shoot and root biomassbetween treatments were tested by one-way analysis of vari-ance (ANOVA) (Tukey). All statistical analyses were con-ducted with SPSS 11.0 (SPSS, Chicago, IL).

Results

Conditions in root containers

At the end of simulated winter period SW2, the water in the or-ganic layer was frozen in all of the treatments (Figure 1), aswas the mineral layer in the treatments where soil thawing hadstarted simultaneously with or following a delay of 14 and24 days after an increase in air temperature (T2, T3 and T4, re-spectively) (data not shown). In contrast, the water content ofthe mineral layer in the treatment involving 14 days of pre-thawing (T1) was still decreasing when the thawing started, in-dicating that freezing was incomplete (data not shown). Soilwater content increased immediately after the soil temperaturewas increased (Figure 1B), with a similar pattern in the organicsoil and mineral soil layers (data for the mineral soil layer isnot shown).

There were no significant differences in soil nutrient con-centrations between the treatments at the end of the study (Ta-ble 1). The pH of the organic layer was lower in T1 and T2 thanin T3 and T4 (mean values of 3.8 and 4.2, respectively) (P <0.05).

Physiology

Treatment, sampling time and their interaction all had a statis-tically significant effect on potential chlorophyll fluorescence(P < 0.001). There was a slight decrease in chlorophyll fluo-rescence (Fv /Fm ratio) in GS1 needles toward the end of SW2in all treatments (Figure 2A). Five days before the start ofGS2, the Fv /Fm ratio was 0.68 in T1, whereas it was close to0.5 in the other treatments; however, the differences were in-significant. When GS2 started, the Fv /Fm ratio increased in T1and T2, but without recovery in T3 and T4. Nine days after thestart of GS2, the Fv/Fm values were significantly lower in T3and T4 than in T1 and T2 (P < 0.01 at maximum). Accordingto the pairwise comparisons, there were no significant differ-ences in the potential chlorophyll fluorescence of the needlesbetween T1 and T2 or between T3 and T4.

Treatment, sampling time and their interaction had signifi-cant effects on the chlorophyll (chl) a/b ratio (P < 0.001). Twoweeks before the start of GS2, the chl a/b ratio of the GS1 nee-dles was about 4 (Figure 2B). There was a slight, insignificantdecrease in the chl a/b ratio toward the end of SW2. The chla/b ratio recovered to close to the initial value in T1 and T2during GS2, but continued to decrease in T3 and T4. By Day31 of the GS2 period, the chl a/b ratio was significantly lowerin T3 and T4 than in T1 and T2 (P < 0.001). According to thepairwise comparisons, there were no significant differences inthe chl a/b ratio between T1 and T2 or between T3 and T4.

Sampling time and its interaction with treatment had a sig-nificant effect on the extracellular resistance of the GS1 nee-dles (P < 0.05 and P < 0.01 respectively). The extracellularresistance ranged from 100 to 110 Ωm during GS1 and SW2(Figure 2C). By Day 10 of GS2, the extracelluler resistancehad fallen below 70 Ωm in T3 and T4 and to about 85 Ωm inT1 and T2. On that occasion, the differences between the treat-ments were insignificant, but thereafter, the extracellular resis-tance strongly increased in T3 and T4. After Day 24 of GS2,

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the extracellular resistance was significantly lower in T1 andT2 than in T4 (P < 0.01). The difference between T1 and T2 orbetween T3 and T4 was not significant on any of the samplingdates tested.

Treatment, sampling time and their interaction had a signifi-cant effect on the water potential of the GS1 needles (P < 0.05,P < 0.01 and P < 0.01, respectively). The water potential of theGS1 needles decreased in T3 and T4 and slightly increased inT1 and T2 at the start of GS2 (Figure 2D). After Day 24 ofGS2, the water potential was significantly lower in T3 and T4

than in T1 and T2 (P < 0.001 and P < 0.01, respectively). Ac-cording to the pairwise comparisons, there was no significantdifference in water potential between T1 and T2 or between T3and T4.

Starch concentration of the GS1 needles increased in T1soon after commencement of GS2, and with some delay in T2(Figure 2E). After a temporary drop in the middle of the grow-ing period, starch concentration continued to increase in bothT1 and in T2. No change in starch concentration occurred inthe saplings in T3 and T4 during GS2. The differences be-

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EFFECTS OF SOIL FROST THAWING ON SCOTS PINE 1057

Figure 2. (A) Potential chloro-phyll fluorescence (Fv /Fm),(B) chlorophyll a/b ratio, (C)extracellular resistance, (D)water potential, (E) starch con-centrations and (F) solublesugar concentrations of theGS1 needles of Scots pine indifferent soil thawing treat-ments T1, T2, T3 and T4 (seearrows in Figures 2A and 2D).Vertical lines indicate cessa-tion and start, respectively, ofthe growing periods GS1 andGS2 with the simulated winterperiod SW2 intervening (seeFigure 1). Time refers to daysfrom the beginning of the ex-periment. Standard error barsare indicated in one directiononly (n = 4).

Figure 3. The mean impedancespectra (imaginary part versusreal part) for stems of Scotspine saplings (n = 4) in soilthawing treatments T1 and T2(A) and T3 and T4 (B). Mea-surements were made 6 weeksafter the start of the growingperiod GS2. In each curve,frequency increases from theright (20 Hz) to left (1 MHz).

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tween treatments were insignificant, but the sampling time ef-fect was significant (P = 0.01).

Sampling time and the interaction with treatment had a sig-nificant effect on the soluble sugar concentration of the GS1needles (P < 0.001 and P < 0.05, respectively); however, nosignificant differences were found between treatments (Fig-ure 2F). Soluble sugar concentration decreased and reached aminimum 57 days after the start of GS2 in T1 and T2, and thenincreased again (Figure 2F).

After 6 weeks of GS2, the impedance spectra in T1 and T2were typical for stems measured by needle electrodes. Thespectra had a long “tail” at low frequencies and an arc at higherfrequencies (Figure 3). In contrast, the shape and magnitude ofthe spectra in T3 and T4 were anomalous, indicating that thestems were drying out.

Shoot and root growth and phenology

All of the saplings in T4 died (Figure 4). A small amount ofgreen foliage remained on some saplings in T3, but no newshoot growth was observed. There was no significant differ-ence in the total biomass of either the shoots or roots betweenT2 and T1 or between T3 and T4 at the end of GS2. However,shoot biomass was lower in T3 and T4 than in T1 (P = 0.07 andP = 0.03, respectively), and somewhat lower in T3 and T4 thanin T2 (P = 0.15 and P = 0.06, respectively) (Figure 4).

The air temperature sum for initiation of apical and lateralshoot elongation, for cessation of the apical shoot elongationand for initiation of needle elongation differed significantlybetween T1 and T2 (P < 0.05) (Table 4). No difference oc-curred in the times of initiation and cessation of stem diametergrowth. The shoot elongation period was approximately thesame for T1 and T2 (in terms of air temperature sum 230 d.d.and 247 d.d., respectively).

Treatment, sampling time and their interaction all had a sig-nificant effect on root-tip formation and total length of themycorrhizas (short roots) and long brown roots (P < 0.01, P <0.001 and P < 0.001 respectively). Root tips formed and rootselongated in T1 at the end of SW2, when the soil had thawedbut air temperature and irradiance were still low (Figure 5). In

T2, root-tip formation and root elongation started soon aftersoil thawing. Thereafter, the rate of root growth did not differbetween T1 and T2. Some new root tips were formed and rootselongated in the saplings of T3 and T4, even though no shootgrowth was observed.

The differences in root growth between T1 and T2 in rela-tion to T3 and T4 gradually increased during GS2. The num-ber of new root tips was significantly higher in T1 than in T3 orT4 already at 15 days, but in T2, it became significant only at57 days after the start of GS2 (P value decreasing from P <0.05 to P < 0.001). Root elongation was significantly higher inT1 than in T4 or T3 at 36 and 51 days after the start of GS2, re-spectively (P value decreasing from < 0.05 to < 0.001). ByDay 50 of GS2, the length of long roots was significantlyhigher in T2 than in T4 or T3 (P value decreasing) from < 0.05to < 0.001). There were no significant differences in root char-acteristics between T1 and T2 or between T3 and T4.

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Figure 4. Shoot biomass of saplings at the beginning of theexperiment (Control), and shoot and root biomass of saplings at theend of the experiment in different treatments. Bars indicate standarderrors for the total biomass of shoots and roots (n = 4). The dead bio-mass of the shoot in T1 and T2 consists of needle litter accumulatedduring the study.

Table 4. Temperature sum (in degree days (d.d.) with 5 °C as a threshold for daily mean temperature) for different phenological events in Treat-ment 1 (T1) and Treatment 2 (T2) (no growth in the other treatments). The running day from the beginning of the experiment for each event is indi-cated in parentheses. The P value indicates the significance of the difference between treatments.

Phenological event T1 (d.d.) T2 (d.d.) P value

Start of diameter growth 110 (186) 76 (184) 0.55Start of shoot elongation of branches 176 (191) 226 (195) 0.02Start of apical shoot elongation 186 (192) 253 (197) 0.04Cessation of shoot elongation of branches 406 (208) 420 (209) 0.77Cessation of apical shoot elongation 416 (209) 500 (215) 0.04Start of needle elongation of apical shoot 443 (211) 486 (214) 0.04Start of needle elongation in branches 446 (211) 460 (212) 0.24Cessation of needle elongation in branches 966 (250) 986 (252) 1.00Cessation of diameter growth 1020 (254) 1050 (257) 0.56Cessation of needle elongation of apical shoot 1050 (257) 1096 (260) 0.66

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Discussion

The start of soil thawing was marked by an increase in soil wa-ter content, and was completed when soil temperature in-creased above 0 °C. In our study, the water content of theorganic layer (Figure 1B) and the mineral soil layer (data notshown) increased immediately once soil thawing began, butsoil temperature increased only after a delay (e.g., 1 week inT2). One explanation for the rapid increase in soil water con-tent during soil thawing is that the ice that had accumulatedduring SW2 on the cooling element set on the soil surfacemelted when the coil temperature rose above 0 °C, releasingwater to the soil. Even though this occurred only once duringthe thawing phase, it may partly mimic the effect of snow meltin spring in the field. Part of the released water percolatedthrough the root container (data not shown), the rest being re-tained by the soil and thus becoming available for uptake byroots. Even though the soil was cool for 1 week after the startof the GS2 period in T2, we conclude that some water uptakeby the roots occurred, because the Fv /Fm ratio returned to itsoriginal value even though needle water potential remainedlower in T2 than in T1 (Figures 2A and 2D).

We found that the timing of soil thawing greatly affectsScots pine saplings. Delayed thawing may cause incipient orchronic injuries. The first symptoms of stress without visibleinjuries were detected in several physiological variables dur-ing the simulated winter period, but they disappeared whensoil water became available in T1 and T2. When the demandfor water increased with increases in air temperature, irradi-ance and photoperiod, the severity of stress and the extent ofvisible injuries increased in the treatments with delayed soilthawing (T3, T4) (Figures 2A–D). As a result, a clear thresh-old appeared between the treatment with a 14-day delay in soilthawing (T3) and the treatment with simultaneous soil and airwarming (T2).

The divergent patterns in needle water potential between thetreatments at the beginning of GS2 indicate that the saplings inT3 and T4 suffered from water stress. In these treatments, nee-dle water potential started to decline as evapotranspirationincreased. The threshold for recovery of needle water potentialwas between –1.5 and –1.7 MPa. Because coniferous seed-lings are known to recover from even lower water potentials(e.g., Havranek and Benecke 1978, Grossnickle 1988), the re-duced water potential was probably not the direct cause of sap-ling death.

During soil thawing, we first observed a decrease in the po-tential efficiency of PSII, as determined by chlorophyll fluo-rescence of dark-adapted needles, and then a decline inchlorophyll a/b ratio. The Fv/Fm ratio decreased slightly evenin T1 where soil thawing started 2 weeks before the onset ofGS2. We conclude that the experimental conditions, i.e.,frozen soil, air temperature of 4 °C and a short (6 h) photo-period with a photon flux of 200 µmol m–2 s–1, induced photo-inhibition in the needles (Sutinen et al. 2000). After the Fv/Fm

ratio had fallen below 0.4, no recovery occurred even thoughsoil water availability improved because of soil thawing. Simi-larly, in white spruce seedlings (Picea glauca (Moench.) Voss)

in controlled freezing tests, visible needle damage appeared atFv/Fm ratios < 0.5 (Gillies and Binder 1997). In the borealzone, however, the Fv/Fm ratio of needles may fall below 0.4 inspring, but recovery may still occur (Westin et al. 1995,Sutinen et al. 2000). Thus, we conclude that the irreversibledamage and death of the needles, e.g., in T3, was not caused bydamage to PSII.

The chl a/b ratio decreased slightly, and then recovered ifthere was no delay or only a short delay in soil thawing (T1 and

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Figure 5. (A) New root-tip formation, (B) elongation of short roots(mostly mycorrhizal) and (C) elongation of long brown roots of Scotspine in different soil thawing treatments as observed with minirhizo-tron tubes set horizontally in the mineral soil layer of the root contain-ers. Arrows in Figure A indicate when the soil thawing started indifferent treatments. Vertical lines indicate cessation and start of thegrowing periods GS1 and GS2, respectively, with the dormancy pe-riod SW2 in between. Time is measured in days from the beginning ofthe experiment. Bars indicate standard errors in one direction only(n = 4).

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T2). If the chl a/b ratio fell below about 2.5 (T3 and T4), no re-covery occurred. The changes in the chl a/b ratio were due tochanges in both chl a and chl b concentrations. At the begin-ning of GS2, chl b concentration tended to increase, whereaschl a concentration remained fairly stable in all treatments(data not shown). After Day 14 of GS2, both the chl a and chl bconcentration continued to rise in T1 and T2, but began to de-cline in T3 and T4. The decline was more delayed in the caseof chl b (start at Day 205) than chl a (Day 190). The increase inboth chl a and b in T1 and T2 (keeping the chl a/b ratio con-stant) indicates the start of assimilation after the beginning ofGS2. In T3 and T4, however, both chl a and chl b weredegraded due to the prolonged water stress, in accordance withprevious studies (Huffaker et al. 1970). Because the chl a/b ra-tio decreased in these treatments, the rupture was more pro-nounced in chl a than in chl b, the former being a centralpigment in PSII. Thus the changes observed in the concentra-tions of chl a and b agree well with the potential efficiency ofthe PSII as determined by chl fluorescence.

The extracellular resistance of needles in T1 and T2, whichwere subjected to a nonlethal stress, was quite stable over thestudy period, whereas changes in T3 and T4 leading to acutedamage were biphasic. During the first 10–15 days after thebeginning of GS2, extracellular resistance first decreased andthen strongly increased. The decrease indicates rupture of cellmembranes and leaching of intracellular ions into the inter-cellular space. A similar phenomenon was observed whenScots pine needles and roots were exposed to frost in con-trolled freezing tests (Repo et al. 1994, Ryyppö et al. 1998). Inour study, cell membrane injuries were probably caused bywater stress. Simultaneously, the photosynthetic machinery ofPSII suffered irreversible damage (cf. Figures 2A and 2C), in-dicating comparable damage to the plasma membrane andthylakoid membranes. The later increase in the extracellularresistance probably reflects drying of the injured needles byevaporation, because this parameter depends strongly on thewater content of the sample (Repo et al. 2002, Luoranen et al.2004).

The IS of stems of healthy saplings in T1 and T2 consistedof one arc at high frequencies and a “tail” at low frequencies.The “tail” is due to the electrode polarization impedance,whereas the arc is due to the stem itself (Repo 1994). Theanomalous IS in T3 and T4 suggest stem tissue injury. Whenthe soil was frozen at the beginning of GS2, it is likely thatevapotranspiration led to embolization of stem vascular tissue(Kramer and Boyer 1995). Living cells of stems and needleswere injured and started to dry out. Two weeks after the start ofGS2, soil water availability increased in T3 (and later in T4),but embolized water conducting cells were not refilled andstem drying continued. Accordingly, the magnitude of boththe real and the imaginary parts of the IS increased (cf. Repo etal. 2002).

Differences between treatments in foliar starch concentra-tion were insignificant. However, the increases in foliar starchconcentration followed the progress of soil thawing: 14 dayspre-thawing (T1) > simultaneous warming of air and soil (T2)

> T3 and T4 (little or no increase), indicating that photosyn-thesis was activated earlier in T1 than in T2, probably a resultof facilitated soil water availability. The characteristic peak ofstarch concentration at the beginning of the growing periodwas lower than in previous studies, indicating either a highsink strength for shoot and root growth, or a low rate of photo-synthesis (Bergh et al. 1998, Oleksyn et al. 2000, Repo et al.2004). In addition, the demand for photosynthates for rootgrowth in T1 was probably already high before the start ofGS2, as indicated by root-tip formation and root elongationand may, therefore, have further dampened the accumulationof starch in needles at the beginning of GS2.

New root-tip formation and elongation of long roots oc-curred during the simulated winter period SW2 in the 14-daypre-thawing treatment (T1), when the soil temperature in-creased to about 10 °C, even though air temperature and irra-diance remained low. Although this root growth may havebeen supported by stored carbohydrates, a likelier cause iscommencement of photosynthesis at the end of SW2, eventhough the prevailing short day lengths may have enhancedmaintenance respiration at the expense of growth (Pelkonen1980, van den Driessche 1987, Bergh and Linder 1999, Suni etal. 2003).

The small differences in the physiological responses be-tween T1 and T2 were not reflected in the biomass differences,nor was the delay in the start of shoot and root growth betweenT1 and T2 reflected in the final biomass. These findings agreewith a study of Scots pine seedlings showing that final root andshoot biomass was unaffected by prolonged periods in coldsoil (5 °C) and warm air (Domisch et al. 2002).

We found that Scots pine saplings can tolerate a few daysdelay in soil thawing after the beginning of the growing sea-son. At this time, transpiration demand may be met by waterstored in the roots and stem sapwood, as occurs in saplings ofEngelmann spruce (Picea engelmannii Parry ex Engelm.)(Boyce and Lucero 1999). In our young saplings, such storagewas probably minimal. To tolerate 14- and 24-day delays insoil thawing (T3, T4), water uptake by the roots would havebeen necessary, but because of the frozen soil this was impos-sible. In a previous study of Engelmann spruce, transpirationdemand and the consequent imposed stress were dependent ontree size (Boyce and Lucero 1999). It was concluded thatsmaller saplings and trees have smaller sapwood water re-serves than larger trees, which means that they rely more onwater that has been stored or absorbed by the roots in order tomeet the demands of transpiration in the winter and spring.This may also be the case for Scots pine, which typically ex-hibits symptoms of winter desiccation at the sapling stage,with the uppermost part barely protruding above the snowcover.

Shoot and root elongation tended to start earlier in the14-day pre-thawing treatment (T1) than in the treatment thatprovided simultaneous soil and air warming (T2). We con-clude, therefore, that if the soil thaws early in the spring, rootfunction will not be the limiting factor in the initiation of shootgrowth. Early soil thaws may already be affecting tree growth

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under present climatic conditions in areas and years with thicksnow cover and shallow soil frost (cf. Solantie 2003). This ef-fect may increase if the climate warms. However, earlier initia-tion of growth may expose shoots to spring frosts common inboreal areas, negating any growth advantage from early soilthawing.

Climate change projections for Finland include increases ina mean annual temperature of 2–7 °C and mean annual precip-itation of 5–40% by 2080 (Jylhä et al. 2004). Most of thechange is predicted to occur in winter and spring, which willprobably affect the depth and duration of the soil frost in bo-real forests (Venäläinen et al. 2001a, 2001b). In response toclimate warming, soil freezing may either increase or de-crease, depending on the quantity and form of precipitation: insome areas where the snow cover currently remains for mostof the winter, it may disappear altogether. Because frosts willprobably not disappear in winter, the depth of soil frost may in-crease in areas with reduced snow cover. However, soil frostmay decrease in areas with increased snow cover. Although wedid not study snow cover and its effects on soil hydrologythrough thawing, our results provide circumstantial evidencethat the annual growth cycle and yield of trees may change inresponse to climate warming, not only because of an increasein air temperature but also because of changes in soil condi-tions.

Acknowledgments

We thank Eija Koljonen, Anita Pussinen, Pekka Järviluoto, UrhoKettunen, Martti Ruha, Leena Karvinen, Merja Niemi, Kati Tanttu,Marjaana Sallinen, Martti Malinen and Vesa Ala-aho for their techni-cal assistance, John Stotesbury for the English revision of the text andUPM-Kymmene Ltd. for providing saplings for the study. The studywas financed by the Finnish Forest Research Institute (Projects 3311and 3394).

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