growth, morphology, and gas exchange in white spruce ( picea glauca )...

Growth, morphology, and gas exchange in whitespruce (Picea glauca) seedlings acclimated todifferent humidity conditions

Jessica J. Roberts and Janusz J. Zwiazek

Abstract: The study examined the effects of different relative humidity conditions at germination, early growth, andfollowing cold storage on morphological and physiological characteristics of white spruce (Picea glauca(Moench)Voss) seedlings. Seedlings that were grown for 18 weeks following seed germination at the lower, 30% RH (RHinitial)treatments were shorter and had smaller stem diameters, shorter needles with more epicuticular wax, and a greater den-sity of needles per centimetre stem, compared with the 80% RHinitial seedlings. After 18 weeks of growth under 30, 50,and 80% RH, the seedlings were hardened off, stored for 8 weeks at 3°C and planted in pots in growth chambers un-der 42 and 74% relative humidity (RHsubsequent). Under 74% RHsubsequentconditions, the lower RHinitial seedlings flushedsooner and had higher growth rates compared with the higher RHinitial seedlings. When the higher RHinitial seedlingswere placed under 42% RHsubsequentconditions, their bud flush was delayed, and subsequent growth rates were lowercompared with the lower RHinitial seedlings. When measured at 40% RH, seedlings subjected to lower RHinitial hadhigher net assimilation rates and stomatal conductance compared with the seedlings acclimated to higher RHinitial hu-midity. It was concluded that the humidity conditions present during early seedling growth following germination sig-nificantly affect their morphological and physiological characteristics during the second growth season.

Résumé: Cette étude porte sur les effets de différentes conditions d’humidité relative au moment de la germination, dela croissance initiale et suivant l’entreposage au froid, sur les caractéristiques morphologiques et physiologiques de se-mis d’épinette blanche (Picea glauca(Moench) Voss). Les semis qui avaient été cultivés pendant 18 semaines suite àla germination des graines au plus bas taux d’humidité (30% HRinitiale) étaient plus petits, avaient une tige de plus petitdiamètre, des aiguilles plus courtes avec plus de cire épicuticulaire et une plus forte densité des aiguilles par centimètrede tige comparativement aux semis soumis au plus haut taux d’humidité (80% HRinitiale). Après 18 semaines de crois-sance à 30, 50 ou 80% HR, les semis étaient endurcis, gardés pendant huit semaines à 3°C, plantés dans des pots etplacés en chambre de croissance à 42 ou 74% d’humidité relative (HRsubséquente). À 74% HRsubséquente, les semis soumisà la plus basse HRinitiale ont débourré plus tôt et avaient un taux de croissance plus élevé que les semis soumis à HRinitiale

la plus élevée. Lorsque les semis soumis à HRinitiale la plus élevée étaient placés à 42% HRsubséquente, leur débourrementétait retardé et le taux de leur croissance subséquente était plus faible comparativement aux semis soumis à la plusbasse HRinitiale. Lorsque les mesures étaient prises à 40% HR, les semis soumis à la plus basse HRinitiale avaient un tauxd’assimilation nette et une conductance stomatale plus élevés que les semis acclimatés à HRinitiale la plus élevée. Nousconcluons que les conditions d’humidité qui prévalent pendant la croissance initiale des semis suite à la germination af-fectent significativement leurs caractéristiques morphologiques et physiologiques pendant la seconde saison de crois-sance.

[Traduit par la Rédaction] Roberts and Zwiazek 1045

Introduction

Although humidity is acknowledged as an important fac-tor regulating the growth and development of plants (Krizeket al. 1971; Morrison-Baird and Webster 1978; Gaffney1978; van de Sanden 1985), few studies have addressed thelonger term effects of humidity on the morphology and

growth of tree seedlings. Even less is known about themechanisms of acclimation in plants to different humidityconditions.

Low atmospheric humidity increases the vapor pressuredeficit (VPD) around plant leaves, which strongly affectsplant water relations. A high water vapor concentration dif-ference between the external atmosphere and internalmesophyll tissues of a leaf causes a high rate of water lossfrom plants to the atmosphere. This may exceed the rate ofwater uptake from the soil and transport through the plant(Gaffney 1978; Nonami et al. 1990; Assmann andGershenson 1991). Excessive transpiration can triggerstomatal closure, which in turn leads to the inhibition ofCO2 uptake and a reduction of photosynthetic rates as wellas water and nutrient transport through a plant (Collatz et al.1991; Weiser and Havranek 1996).

Can. J. For. Res.31: 1038–1045 (2001) © 2001 NRC Canada

1038

DOI: 10.1139/cjfr-31-6-1038

Received August 22, 2000. Accepted February 6, 2001.Published on the NRC Research Press Web site on June 5,2001.

J.J. Roberts and J.J. Zwiazek.1 Department of RenewableResources, University of Alberta, 4-42 Earth SciencesBuilding, Edmonton, AB T6G 2E3, Canada.

1Corresponding author (e-mail: [email protected]).

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Humidity plays a crucial role in the nursery production ofconifer seedlings (Landis et al. 1992). High humidity levelscan cause excess water vapor to condense on seedlings orcontainer walls, providing conditions for the growth of op-portunistic and pathogenic organisms, while low humidityimposes water stress on seedlings (Landis et al. 1989, 1992,1994; Elad et al. 1996). White spruce (Picea glauca(Monech) Voss) seedlings reared in tree nurseries undergo atleast five distinct growth phases including germination, earlygrowth, rapid (exponential) growth, bud initiation, and stemfinishing. Traditionally, moderately high to high humiditylevels are maintained during the germination, early and rapidgrowth phases of white spruce seedlings, to reduce thetranspirational demand during shoot elongation and needleformation (Wood 1995; Landis et al. 1992). However, sinceduring these growth phases most of the new seedling tissuesare formed, this practice produces seedlings that are accli-mated both morphologically and physiologically to lowtranspirational demand conditions. Such high humidity nurs-ery conditions are rarely found on harvested forest sites,which are often characterized by increased incidence ofwind gusts and large temperature and humidity fluctuations(Balisky and Burton 1995; Jordan and Smith 1995; Marsdenet al. 1996; Kimmins 1997; Man and Lieffers 1997). Sincefew studies have focused on humidity as a factor in coniferseedling growth, the importance of humidity in preventingpost-planting shock and mortality has been often overlooked.

Morphological and physiological characteristics of plantsvary in response to the environmental conditions underwhich they are grown (Zwiazek and Blake 1989; Leverenzand Hinckley 1990; Reich et al. 1996; Sprugel et al. 1996;Man and Lieffers 1997). Therefore, it is possible that seed-lings may acclimate to the humidity levels under which theydeveloped in the greenhouse and that this acclimation re-sponse may be used to produce seedlings with desirableplanting characteristics.

In the present study, we hypothesized that, in contrast tohigh humidity-grown seedlings, plants developed under low

humidity (high transpirational demand) acquire morpho-logical and physiological characteristics that allow them totolerate both low and high humidity conditions. The objec-tives of the present work were to (i) examine the effects ofrelative humidity on the growth, morphology, and gas ex-change of white spruce seedlings and (ii ) determine howseedlings grown under different humidity conditions respondto humidity treatments during their second growth season.

Materials and methods

Germination, early and rapid growth phasesWhite spruce seeds from Slave Lake, Alberta, Canada (seedlot

No. DS 70-9-5-91, elevation 670 m) were used in this study. Apeat–vermiculite (3:1 by volume) soil mixture was adjusted to apH of 5.5 with dolomite lime and autoclaved at 160°C for 60 min.Nine Styroblock trays with 60-mL cavities (Beaver Plastics, Ed-monton, Alta., Canada) were sterilized and filled with the soil mix-ture to the recommended density of 0.09 g/mL (Wood 1995). Threeseeds per cavity were sown but thinned to one seedling per cavityafter germination. This would produce 1440 experimental seed-lings.

To germinate, the seeded Styroblocks were randomly placed inthree growth chambers (three Styroblocks per chamber) and main-tained under the germination conditions specified in Table 1. Aftergermination was complete (end of week 2), the growth chamberswere set to the early growth phase environmental conditions(Landis et al. 1989; Table 1), until the seedlings had clearly be-come established. At 5 weeks (still the early growth phase of seed-ling development), the Styroblocks were once again rearrangedbetween chambers, and the three experimental humidity conditionswere introduced as outlined in Table 1. Seedlings experienced theirexponential growth phase between weeks 10 and 18 and were stillunder the experimental humidity conditions.

To determine watering frequency, Styroblock masses before wa-tering (dry-down masses), soil pH, and soil electrical conductivitymeasurements were made every 3 or 4 weeks and adjusted as nec-essary to remain within recommended guidelines (Wood 1995).Seedlings were fertilized using a constant feed program accordingto the growth stage (Wood 1995) and rearranged within their

© 2001 NRC Canada

Roberts and Zwiazek 1039

Growth phaseDuration(weeks)

Air temperature (°C) PAR(µmol·m–2·s–1)

Photoperiod(h) RH (%)Day Night

Germination 0–3 20.0±2.2 18 380±20 18 78±5.3Early growth 3–10 19.9±1.2 18 375±35 18 31.0±2.8a

48.7±3.3b

80.4±0.9c

Rapid growth 10–19 20.0±0.98 18 375±27 18 31.0±2.8a

48.7±3.3b

80.4±0.9c

Bud initiation 19–21 12.0±1.1 10 380±18 14 33.1±4.0a

47±8.6b,c

Stem finishing 21–24 12.0±1.1 10 370±31 14 33.1±4.0a

47±8.6b,c

Subsequent growth 8 21.0 18 385±25 18 42.2±2.2a

8 21.0 18 390±10 18 73.7±3.4c

Note: Experimental conditions were introduced during week 5.aLow-humidity growth chamber.bModerate-humidity growth chamber.cHigh-humidity growth chamber.

Table 1. Measured values of air temperature, photosynthetically active radiation (PAR), photoperiod,and relative humidity (RH) in growth chambers during various stages of seedling development.



respective growth chambers. Temperature and humidity valueswere recorded from sensors located within the growth chambers,and were compared with measurements from thermometers, whichwere placed on top of the Styroblocks and left within the cham-bers. In addition, the humidity was monitored weekly with a slingpsychrometer.

Experimental treatmentsThe three growth chambers used in this study were maintained

at the same environmental conditions with the exception of the rel-ative humidity (RH) treatment. At the beginning of week five,growth chambers were set to 80, 50, or 30% RH (RHinitial). Seed-lings were grown under these experimental RH conditions halfwaythrough their early growth phase, through the exponential growthand budset initiation phases, until week 21 (the beginning of thestem finishing phase), for a total of 16.5 weeks. Table 2 convertstargeted RH treatments to vapor pressure deficit (VPD). VPD is be-lieved to be the humidity parameter detected by plant guard cells(Assmann and Gershenson 1991) and is often used to determineevapotranspirational demand on plants. Container tree nurseriesrecommend maintaining the VPD below 1.0 kPa (Landis et al.1989).

Seedling hardeningSeedlings were removed from the experimental treatments dur-

ing week 21. At that time, growth chamber conditions were alteredto inhibit seedling shoot elongation and induce bud set (Table 1). Itis recommended that the high humidity be reduced during the budinitiation and stem finishing growth phases to initiate the hardeningprocess (Landis et al. 1989; Wood 1995). Therefore, at weeks 21,the 80% RHinitial seedlings were placed under 50% RH, while theRH conditions from the 30 and 50% RHinitial treatments remainedunchanged. By week 24, the seedlings had hardened off and wereremoved from the Styroblocks, bundled into groups of 20, and thesoil plugs wrapped in plastic. Bundles were placed in wax-linedboxes and stored at 3 ± 1°C (Macey and Arnott 1986; van denDriessche 1991) for 8 weeks before further testing.

Growth measurementsShoot height was measured on 45 randomly selected seedlings

from each RHinitial treatment approximately every 2 weeks, begin-ning 6 weeks after sowing. Measurements were made from the topof the soil plug to the tips of the terminal needles (Thompson1985). Stem diameter measurements commenced after week 10and were made approximately 0.5 cm above the soil plugs(Thompson 1985).

Epicuticular wax contentOnce seedlings reached 26 weeks, 10 seedlings from each

RHinitial treatment were randomly selected, and the shoots rinsedwith deionized water and immersed in liquid nitrogen to facilitateremoval of needles. The needles were then freeze-dried and stored

at –20°C until the time of wax analysis. To quantify theepicuticular wax content, approximately 20 mg (13–18 needles)from each seedling were weighed and placed in a glass test tube.Chloroform (3.0 mL) was added to the tube (Gulz 1994), and thetube was vortexed for 30 s to chemically remove the epicuticularwax. The chloroform was decanted through two layers of cheese-cloth, and the needles were dried for 4 days then reweighed. Thedifference between initial and final masses represented the quantityof epicuticular wax removed.

Bud flush under subsequent RH conditionsThirty RHinitial seedlings from each treatment were removed

from cold storage and planted in 4-L pots (three seedlings per pot,one from each RHinitial treatment,) containing peat–vermiculite (3:1by volume). All pots were placed at the humidity set to 42%(RHsubsequent) (measured RH 42.2 ± 2.2), 21:18°C, 18 h light : 6 hdark photoperiod (PAR approximately 390µmol·m–2·s–1). This pro-cess was repeated with another group of 30 RHinitial seedlings, onlythese pots were placed at 74% RHsubsequent(measured RH 73.7 ±3.4%) with other conditions similar to the 42% RHsubsequenttreat-ment (Table 2). The purpose of this was to flush and grow theRHinitial seedlings under second-year growth conditions of lower(42%) or higher (74%) RH. All seedlings were monitored every3 days for signs of terminal and lateral bud flush. Bud flush wasdefined as having at least 1.0 mm of new needle tissue exposed andwas recorded for all 60 seedlings. Results were recorded as the cu-mulative percentage of seedlings within a treatment that hadflushed their terminal buds, or at least one lateral bud, from thetime of potting.

Shoot and needle morphologyMorphological measurements, including shoot height, needle

length, and needle density were made on 15 seedlings from threegroups: (i) RHinitial seedlings immediately removed from cold stor-age; (ii ) RHinitial seedlings removed from cold storage and grownfor 8 weeks under 42% RHsubsequent; and (iii ) RHinitial seedlingsremoved from cold storage and grown for 8 weeks under 74%RHsubsequent. All needle measurements from the RHsubsequenttreat-ments were made on new tissue growth. Needle density was de-fined as the number of needles present within a 1.0-cm section ofstem. All measurements were made using fully expanded matureneedles located at the midpoint along the main stem. Because of asampling error, some values are unavailable for the 42% RHsubsequentseedlings.

Gas exchangePhysiological measurements were conducted on the RHinitial

seedlings removed from cold storage, potted, flushed, and grownunder 42% and 74% RHsubsequentconditions as described above.Net assimilation at light saturation (Amax) and stomatal conduc-tance (gs) were measured on new growth tissues 12 and 17 days af-ter potting. Six seedlings per treatment were measured on eachdate, with four measurements averaged over a 2-min time period.All readings were made at 30-s intervals after photosynthetic rateshad stabilized, with the humidity in the conifer cuvette set to 40%RH and CO2 set to 350 ppm. Gas-exchange measurements weremade using an LCA-4 infrared gas analyzer (IRGA; Analytical De-velopment Company Ltd., Hoddesdon, U.K.) fitted with a conifercuvette. For gas-exchange measurements, seedlings were removedfrom the growth chambers and acclimated for approximately 5 minto the following measurement conditions: temperature 22 ± 1°C,RH 40 ± 2%, and the photosynthetically active radiation (PAR)supplemented with a quartz halogen lamp to approximately1000 µmol·m–2·s–1. Projected leaf areas were determined fromscanned images of the needles and calculated using Sigma ScanPro 3.0 software from Jandal Scientific (San Rafael, Calif.).

© 2001 NRC Canada

1040 Can. J. For. Res. Vol. 31, 2001

Temperature (°C) RH (%) VPD (kPa)

Treatment Day Night Day Night Day Night

30% RHinitial 20 18 30 30 1.63 1.44

50% RHinitial 20 18 50 50 1.17 1.03

80% RHinitial 20 18 80 30 0.47 0.41

42% RHsubsequent 21 18 42 42 1.43 1.19

74% RHsubsequent 21 18 74 74 0.65 0.54

Table 2. Preset temperature, relative humidity (RH), and vaporpressure deficit (VPD) values for the RHinitial and RHsubsequent

treatments.



Data analysisAll data were analyzed for treatment significance using an

ANOVA, and between treatment means using the Tukey’sstudentized range test (SAS statistical software package, version6.11; SAS Institute Inc., Cary, N.C.).

Results

Initial seedling growthAfter 3 weeks of humidity treatments, 30 and 50% RHinitial

seedlings showed significantly lower height growth (p =0.04) compared with seedlings from the 80% RHinitial treat-ment (Fig. 1a). At week 14, significant height differenceswere apparent between the 30 and 50% RHinitial seedlings.Treatment effects on stem diameter followed the same gen-eral trend as the height data (Fig. 1b). However, significant(p = 0.02) differences in stem diameter between the 80%and the 30% and 50% RHinitial seedlings were not apparentuntil week 12. No differences in stem diameter were foundbetween the 30 and 50% RHinitial.

Epicuticular waxSeedlings grown under 80% RHinitial conditions produced

needles with a significantly greater dry mass compared withthose under 50% RHinitial, but with less epicuticular waxcompared with 30% RHinitial seedlings (Table 3). Seedlingsreared under lower humidity conditions contained needleswith greater quantities of epicuticular wax, and these differ-ences were statistically significant between 30% RHinitial and80% RHinitial treatments (Table 3).

Bud flush under subsequent RH conditionsSeedlings from all RHinitial treatments flushed sooner un-

der 74% compared with 42% RHsubsequent(Fig. 2a). In gen-eral, 30 and 50% RHinitial seedlings flushed their terminalbuds earlier than seedlings from the 80% RHinitial treatments(Fig. 2a). Under 74% RHsubsequentconditions, all seedlingsfrom the 30 and 50% RHinitial treatments had flushed theirterminal buds by day 18, while those from the 80% RHinitialtreatment required an additional 6 days (Fig. 2a).

Lateral bud flush under the 74% RHsubsequentconditionsfollowed the same general trend as that of the terminal buds(Fig. 2b). Seedlings from all three RHinitial treatmentsshowed signs of lateral bud flush by day 9, but again, agreater proportion of flushed seedlings were from the 30%RHinitial treatment, followed by the 50 and 80% RHinitialtreatments. By day 15, all 30 and 50% RHinitial seedlings hadopened at least one lateral bud, but seedlings from the 80%RHinitial treatment required an additional 3 days. Under the42% RHsubsequentconditions, lateral bud flush occurred for allthree RHinitial treatments by day 9, three days later comparedwith seedlings flushed under 74% RHsubsequent.

Shoot and needle morphologyIn the subsample of RHinitial seedlings removed from cold

storage, we found no differences in height between the 30and 50% RHinitial seedlings, but the 80% RHinitial seedlingswere significantly taller (Table 4). The 80% RHinitial seed-lings showed a greater stem diameter compared with the 30and 50% RHinitial seedlings (Table 4).

After growing these seedlings for 8 weeks under 74%RHsubsequent conditions, seedlings from all three RHinitialtreatments showed similar final heights. However, there weresignificant (p < 0.001) treatment effects on both the lengthof new shoot growth, and the new:old shoot ratio. The 30%RHinitial seedlings showed an almost 53% increase in shootheight compared with a 41% increase for 50% and a 26% in-crease for 80% RHinitial seedlings (Table 4). Most of this in-crease was due to new shoot growth. After 8 weeks ofgrowth under 74% RHsubsequent, the 30% RHinitial seedlingshad significantly smaller stem diameters (p = 0.040) com-pared with the 50 and 80% RHinitial seedlings (Table 4). Be-cause of a sampling error, shoot heights under 42%RHsubsequentare unavailable.

For all RHinitial seedlings, needle length significantly (p <0.001) increased with increasing higher relative humidity ex-

© 2001 NRC Canada


Needle drymass (mg)

Epicuticular wax(mg·g–1 DM)

30% RH 1.3 (0.3)ab 16.2 (2.9)a50% RH 1.0 (0.2)a 14.3 (3.3)ab80% RH 1.5 (0.4)b 12.8 (1.83)b

Note: Measurements were made after seedlingswere hardened off but before cold storage. Valuesare means with SE given in parentheses. Values withdifferent letters within a column are significantlydifferent (p = 0.05) based on the Tukey’s test (n =10). DM, dry mass.

Table 3. Mean needle dry mass andepicuticular wax content in white spruce seed-lings grown under the three RHinitial conditions.

2

6

10

14

18

22

Heig

ht

(cm

)

30% RH

50% RH

80% RH

a

0

1

2

3

4

6 8 10 12 14 16 18 20 22 24 26

Weeks after sowing

Ste

md

iam

ete

r(m

m)

b

Fig. 1. Height (a) and stem diameter (b) of white spruce seed-lings from the 30, 50, and 80% relative humidity (RH) treat-ments. Means ± SE are shown (n = 45).



posure (Table 5). However, after RHinitial seedlings weregrown under 42% RHsubsequent, the reverse trend was ob-served, with the 80% RHinitial seedlings producing signifi-cantly shorter needles compared with the 30 and 50%RHinitial seedlings. After flushing under 74% RHsubsequentconditions, no differences in needle length were found be-tween the RHinitial seedlings.

Originally, needle density for the 30 and 50% RHinitialseedlings was significantly (p = 0.0002) higher comparedwith those from 80% RHinitial treatment (Table 5). Afterflushing under 42% and 72% RHsubsequent, the reverse trendwas seen, with the 30 and 50% RHinitial seedlings producingshoots with significantly (p < 0.001) lower densities than the80% RHinitial seedlings (Table 5).

Gas exchangeAfter growing seedlings from the three RHinitial treatments

under 74% RHsubsequentconditions, no differences inAmaxvalues were found (Fig. 3a). Small but significant (p = 0.046)treatment differences inAmax were found between the threeRHinitial seedlings that were grown under 42% RHsubsequent.The 30% RHinitial seedlings maintained the highest values,followed by the 50 and 80% seedlings (Fig. 3a). Althoughall gas-exchange measurements were conducted under a hu-midity level of 40 ± 2%,Amax rates were significantly higherin all RHinitial seedlings grown under 74% compared with42% RHsubsequentconditions (Fig. 3a).

When grown under 42% RHsubsequent, the 30 and 50%RHinitial seedlings had significantly highergs values com-pared with the 80% RHinitial seedlings (Fig. 3b). However,

when measured on day 17, the 80% RHinitial seedlings hadthe highestgs values.

Discussion

Humidity treatments profoundly affected the growth andmorphology of white spruce seedlings. Low shoot growthrates observed in the low and moderate humidity grownseedlings could be partly explained by stomatal factors.Plants respond to high transpirational water loss by closingtheir stomata, even if there is an adequate water supply tothe roots (Larcher 1995; Percival et al. 1996). In turn,stomatal closure can lead to a decrease in rates of photosyn-thesis and transpiration (Nonami et al. 1990; Assmann andGershenson 1991; Weiser and Havranek 1996). In our study,the 30 and 50% RHinitial seedlings were shortest after initialgrowth. However, subsequent growth under 74% RH re-sulted in higher terminal shoot elongation rates comparedwith the 80% RHinitial seedlings. This suggests that the long-term morphological and (or) physiological characteristicsproduced in seedlings initially acclimated to low RH werenot carried over into the next growing season and, in fact, al-lowed the seedlings to take advantage of subsequent, higherRH conditions to promote shoot elongation. Since physio-logical measurement were not made on seedlings growingunder the initial three humidity conditions, it is unclear if theseedlings maintained similar photosynthetic rates, andphotosynthate was stored rather that directed to shoot elon-gation. Future studies on carbohydrate stores on low humid-ity reared seedlings would help answer these questions.

The 30% RHinitial seedlings produced shorter needles thatwere more densely spaced compared with the higher RHinitialgrown plants. However, similar to seedling growth, whenthese plants where exposed in the next growing season to42% RHsubsequentconditions, they produced longer needlescompared with those acclimated to 50 or 80% RHinitial. Thisindicates that the humidity conditions before bud set can af-fect the length of needles in the subsequent year. It has beengenerally accepted that leaf length varies depending on envi-ronmental conditions at the time of leaf expansion (Nobel1980). Our study supports this view since white spruce seed-lings produced longer needles when exposed to higher RH atthe time of leaf elongation. However, we also demonstratedthat environmental conditions prior to bud set in whitespruce can have a profound effect on the elongation pro-cesses taking place in the following year. As with the growthof shoots, it appears that this response was likely due tophysiological and morphological changes at low humidityconditions.

It is plausible that the morphological and physiologicalcharacteristics similar to those that affected height growthplayed a role in altering needle length in seedlings raised inlow humidity when subsequently exposed to higher humidityconditions. Such features could affect water relations underthe new humidity conditions to facilitate growth processesand may have included reduced stomatal sensitivity, in-creased plant hydraulic conductivity, and increased water useefficiency (Zwiazek and Blake 1989; Wan and Zwiazek1999). However, to explain plant response to RH followingcold storage and bud flushing, these characteristics wouldhave to persist for at least several months. Among the stud-

© 2001 NRC Canada

1042 Can. J. For. Res. Vol. 31, 2001

0

20

40

60

80

100

%S

ee

dli

ng

s

a (terminal buds)

0

20

40

60

80

100

3 6 9 12 15 18 21 24

Days after planting

%S

ee

dli

ng

s

30:74% RH

50:74% RH

80:74% RH

30:42% RH

50:42% RH

80:42% RH

b (lateral buds)

Fig. 2. Time of flushing of the terminal buds (a) and lateralbuds (b) in seedlings reared under the 30, 50, and 80% relativehumidity (RH) treatments and placed under 74 and 42% relativehumidity (n = 30).



ied morphological characteristics produced as a result of lowRH in growing seedlings, it is unlikely that high needle den-sity, short needles, and increased epicuticular wax contentcould contribute to higher growth rates of these seedlingsunder a relatively high RH. However, it is plausible thatother structural changes may have played a role in thisgrowth response. The 50% RH seedlings had the lowestmass per needle but had significantly longer needles com-pared with the 30% RH seedlings. This suggests that lowerhumidity levels may have produced structural modificationsin the needles.

Chloroform-soluble waxes represent the greatest barrier totranscuticular water vapor diffusion (Hadley and Smith1990). Wax quantity per gram of dried needle tissue washigher in the 30% compared with the 80% RH needles, butvalues were within similar ranges reported for Norwayspruce (Picea abies (L.) Karst.) needles by Guenthardt(1984) and Cape and Percy (1993). Since large differencesin wax quantity were not found between the three treat-ments, it is unlikely that low humidity levels per se induceda greater quantitative production of epicuticular wax inwhite spruce seedlings. However, low humidity might haveaffected the composition and structure of the epicuticularwaxes, altering their physical properties such as permeabilityto water vapor diffusion.

In the present study, the lower 42% RH level delayed theonset of lateral and terminal bud flush by approximately 3–9 days compared with 74% RH. A similar effect of RH onbud flush in white spruce was reported earlier (Mardsen etal. 1996). Our present results indicate that the humidity lev-els present before bud set can also affect the timing of budflush. Lavender (1985) acknowledged soil moisture,photoperiod, and temperature as major factors that can con-

© 2001 NRC Canada


Hardenedseedlings

74% RHsubsequent

flushed seedlings New growth (cm)New growthincrease (%)

Height (cm)30% RH 16.7 (1.1)a 29.5 (1.4)a 15.7 (1.1)a 52.9 (1.7)a50% RH 18.7 (1.7)a 28.6 (1.1)a 11.8 (0.8)b 40.9 (2.1)b80% RH 25.3 (2.3)b 30.5 (1.4)a 8.06 (1.1)c 26.0 (2.3)cDiameter (mm)30% RH 2.96 (0.1)a 5.17 (0.2)a 2.21a 40.9a50% RH 3.05 (0.1)a 5.71 (0.2)b 2.66a 45.1a80% RH 3.37 (0.1)b 5.91 (0.2)b 2.54a 41.9a

Note: Measurements are from a subsample of RHinitial seedlings immediately removed from coldstorage, while flushed seedlings refers to those grown under 74% RHsubsequentand measured after 8weeks. Values are means with SE given in parentheses. Values with the same letter within a column arenot statistically different at the 0.05 level based on a Tukey’s test (n = 30).

Table 4. Comparison of shoot height and stem diameter of seedlings from the three hu-midity treatments.

Hardenedseedlings

42% RHsubsequent

flushed74% RHsubsequent

flushed

Needle length (cm)30% RH 1.65 (0.1)a 2.12 (0.1)a 2.18 (0.1)a50% RH 1.96 (0.1)b 1.93 (0.1)a 2.13 (0.1)a80% RH 2.21 (0.1)c 1.60 (0.1)b 1.98 (0.1)aNeedle density (needles/cm stem)30% RH 33.0 (1.8)a 24.7 (1.5)a 16.7 (1.1)a50% RH 29.5 (1.1)a 27.9 (2.1)a 18.7 (1.4)a80% RH 23.5 (1.2)b 34.3 (2.0)b 25.3 (2.2)b

Note: Values are means with SE given in parentheses. Values with thesame letter within a column are not statistically different at the 0.05 levelbased on a Tukey’s test (n = 30).

Table 5. Needle length and density in seedlings from the threehumidity treatments before and after subjecting them to 42 and74% RHsubsequent.

0

2

4

6

8

10

12

30:42% RH 50:42% RH 80:42% RH 30:74% RH

50:74% RH 80:74% RH

a

a

b

c

a

a

b

c

a

a

a

0

100

200

300

400

500

600

700

800

12 17

Days after planting

b

a a b

a

b

b

a b c

a

b

b

A(

mol

CO

·m·s

max

2-2

-

1µ

)g

(mm

ol·m

·ss

-2

-1 )

Fig. 3. Net assimilation (a) and stomatal conductance (b) inseedlings reared under the 30, 50, and 80% relative humidity(RH) treatments hardened off, potted, and placed for 12 and17 days under 74 and 42% relative humidity. Bars are means(n = 6), and bars are SE. Different letters above the bars withinthe same treatment group indicate statistically significant differ-ences atp = 0.05 determined by the Tukey’s test.

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tribute to delayed seedling bud flush. However, our earlierstudies (Marsden et al. 1996; Wang and Zwiazek 1999a,1999b, 1999c), unpublished studies, and the present work in-dicate that additional factors that affect plant water relations,including relative humidity, winter storage temperature andsoil temperature, influence the time of bud flush in whitespruce seedlings. This response can be probably explainedby the demand of expanding leaf and stem cells for water.

Net assimilation and stomatal conductance were reducedin seedlings growing under the lower, compared with thehigher, RH for all treatments. Similar results have been re-ported for several plant species including white spruce (Daiet al. 1992; Stewart and Bernier 1995; Marsden et al. 1996;Percival et al. 1996; Zhang et al. 1996). Under 74% RH con-ditions, stomatal conductance was significantly lower in the30% RH seedlings, while net assimilation was similar to theremaining RH treatments. Under the 42% RH conditions, the30% RH seedlings had higher net assimilation and stomatalconductance compared with the 50 and 80% RH seedlings.These results indicate that, compared with nonacclimatedseedlings, the seedlings that have been acclimated to low RHconditions can reach higher photosynthetic rates when sub-sequently exposed to higher RH.

In our study, white spruce seedlings that developed under30% RH were able to tolerate the relatively low, 42% RHbetter than the seedlings that were grown under higher RH.This has important practical implications for reforestationprograms, since low RH conditions may be expected inmany white spruce planting sites (Marsden et al. 1996; Manand Lieffers 1997).

Acknowledgments

We gratefully acknowledge financial support for this studyfrom the Pine Ridge Forest Nursery (Smoky Lake),Weyerhaeuser Canada (Grande Prairie), Manning DiversifiedForest Research Trust Fund, and Natural Sciences and Engi-neering Research Council of Canada in the form of researchgrant to J.J.Z. as well as the University of Alberta DesmondCrossley Memorial and the Max Mclaggan scholarships toJ.J.R.

References

Assmann, S.M., and Gershenson, A. 1991. The kinetics of stomatalresponse to VPD inVicia faba: electrophysiological and waterrelations models. Plant Cell Environ.14: 455–465.

Balisky, A.C., and Burton, P.J. 1995. Root-zone soil temperaturevariation associated with microsite characteristics in high-elevationforest openings in the interior of British Columbia. Agric. For.Meteorol.77: 31–54.

Cape, J.N., and Percy, K.E. 1993. Environmental influences on thedevelopment of spruce needle cuticles. New Phytol.125: 787–799.

Collatz, G.J., Ball, J.T, Grivet, C, and Berry, J.A. 1991. Physiologi-cal and environmental regulation of stomatal conductance, pho-tosynthesis and transpiration: a model that includes a laminarboundary layer. Agric. For. Meteorol.54: 107–136.

Dai, Z., Edwards, G.E., and Ku, M.S.B. 1992. Control of photo-synthesis and stomatal conductance inRicinus communisL.(castor bean) by leaf to air vapor pressure deficit. Plant Physiol.99: 1426–1434.

Elad, Y., Malathrakis, N.E., and Dik, A.J. 1996. Biological controlof Borytris-incited diseases and powdery mildews in greenhousecrops. Crop Protect.15: 229–240.

Gaffney, J.J. 1978. Humidity: basic principles and measurementtechniques. HortScience,13: 551–555.

Guenthardt, M.S. 1984. Epicuticular wax ofPicea abiesneedles.In Structure, function and metabolism of plant lipids.Edited byP.A. Siegenthalar and W. Eichenberger. Elsever Science, NewYork. pp. 499–502.

Gulz, P.G. 1994. Epicuticular wax in the evolution of plant king-dom. J. Plant Physiol.143: 453–464.

Hadley, J.L., and Smith, W.K. 1990. Influence of leaf surface waxand leaf area to water content ratio on cuticular transpiration inwestern conifers. Can. J. For. Res.20: 1306–1311.

Jordan, D.N., and Smith, W.K. 1995. Microclimate factors influ-encing the frequency and duration of growth season frost forsubalpine plants. Agric. For. Meteorol.77: 17–30.

Kimmins, J.P. 1997. Balancing act: environmental issues in for-estry. University of British Columbia Press, Vancouver.

Krizek, D.T., Bailey, W.A., and Klueter, H.H. 1971. Effects of rela-tive humidity and type of container on the growth of F1 hybridannuals in controlled environments. Am. J. Bot.58: 544–551.

Landis, T.D., Tinus, R.W., McDonald, S.E., and Barnett J.P. 1989.Seedling nutrition and irrigation. Vol. 4. The container tree nurs-ery manual. U.S. Dep. Agric. Agric. Handb. 674.

Landis, T.D., Tinus, R.W., McDonald, S.E., and Barnett J.P. 1992.The container tree nursery manual. Vol. 3. Atmospheric environ-ment. U.S. Dep. Agric. Agric. Handb. 674.

Landis, T.D., Tinus, R.W., McDonald, S.E., and Barnett J.P. 1994.The container tree nursery manual. Vol. 1. Nursery planning, de-velopment and management. U.S. Dep. Agric. Agric. Handb.674.

Larcher, W. 1995. Physiological plant ecology. Springer-Verlag,New York.

Lavender, D.P. 1985. Bud dormancy.In Evaluating seedling qual-ity: principles, procedures and predictive abilities of major tests.Edited by M.L. Duryea. Forest Research Laboratory, OregonState University, Corvallis. pp. 7–15.

Leverenz, J.W., and Hinckley, T.M. 1990. Shoot structure, leaf areaindex and productivity of evergreen conifer stands. Tree Physiol.6: 135–150.

Macey, D.E., and Arnott, J.T. 1986. The effect of moderate mois-ture and nutrient stress on bud formation and growth ofcontainer-grown white spruce seedlings. Can. J. For. Res.16:949–954.

Man, R., and Lieffers, V.J. 1997. Seasonal photosynthetic re-sponses to light and temperature in white spruce (Picea glauca)seedlings planted under an aspen (Populus tremuloides) and inthe open. Tree Physiol.17: 437–444.

Marsden, B.J., Lieffers, V.J., and Zwiazek, J.J. 1996. The effect ofhumidity on photosynthesis and water relations of white spruceseedlings during the early establishment phase. Can. J. For. Res.26: 1015–1021.

Morrison-Baird, L.A., and Webster, B.D. 1978. Relative humidityas a factor in the structure and histochemistry of plants.HortScience,13: 8–10.

Nobel, P.S. 1980. Leaf anatomy and water use efficiency.In Adap-tations of plants to water and high temperature stress.Edited byN.C. Turner and P.J. Kramer. John Wiley, New York. pp. 43–55.

Nonami, H., Schulze, E.D., and Ziegler, H. 1990. Mechanisms ofstomatal movement in response to air humidity, irradiance andxylem water potential. Planta,183: 57–64.

Percival, D.C., Proctor, J.T.A., and Tsujita, M.J. 1996. Whole plantnet CO2 exchange of raspberry as influenced by air and root-

© 2001 NRC Canada

1044 Can. J. For. Res. Vol. 31, 2001



© 2001 NRC Canada


zone temperature, CO2 concentration, irradiation and humidity.J. Am. Soc. Hortic. Sci.121: 838–845.

Reich, P.B., Oleksyn, J., Modrzynski, J., and Tjoelker, M.G. 1996.Evidence that longer needle retention of spruce and pine popula-tions at high elevations and high altitudes is largely aphenotypic response. Tree Physiol.16: 643–647.

Sprugel, D.G., Brooks, J.R., and Hinckley, T.M. 1996. Effects oflight on shoot geometry and needle morphology inAbiesamabilis. Tree Physiol.16: 91–98.

Stewart, J.D., and Bernier, P.Y. 1995. Gas exchange and water rela-tions of 3 sizes of containerizedPicea marianaseedlings sub-jected to atmospheric and edaphic water stress under controlledconditions. Ann. Sci. For.52: 1–9.

Thompson, B.E. 1985. Seedling morphological evaluation-whatyou can tell from looking.In Evaluating seedling quality: princi-ples, procedures and predictive abilities of major tests.Edited byM.L. Duryea. Forest Research Laboratory, Oregon State Univer-sity, Corvallis. pp 59–71.

van de Sanden, P.A.C.M. 1985. Effects of air humidity on growthand water exchange of cucumber seedlings. Preliminary report.Acta Hortic. 174: 259–264.

van den Driessche, R. 1991. Influence of container nursery regimeson drought resistance of seedlings following planting. I. Sur-vival and growth. Can. J. For. Res.21: 555–565.

Wan, X., and Zwiazek, J.J. 1999. Mercuric chloride effects on rootwater transport in aspen seedlings. Plant Physiol.121: 939–946.

Wang, Y., and Zwiazek, J.J. 1999a. Spring changes in water rela-tions, gas exchange, and carbohydrates of white spruce (Piceaglauca) seedlings. Can. J. For. Res.29: 332–338.

Wang, Y., and Zwiazek, J.J. 1999b. Effects of storage temperatureon physiological characteristics of fall-lifted white spruce (Piceaglauca) bareroot seedlings. Can. J. For. Res.29: 679–686.

Wang, Y., and Zwiazek, J.J. 1999c. Effects of early spring photo-synthesis on carbohydrate content, bud flushing and root andshoot growth ofPicea glaucabareroot seedlings. Scand. J. For.Res.14: 295–302.

Weiser, G., and Havranek, W.M. 1996. Evaluation of ozone impacton mature spruce and larch in the field. J. Plant Physiol.148:189–194.

Wood, B. 1995. Conifer seedling grower guide. Alberta Environ-mental Protection, Smoky Lake.

Zhang, Y., Xu, X.L., Dansereau, B., and Gosselin, A. 1996.Photosynthetic response of hibiscus plants to short-term humid-ity change. Biotronics,25: 23–31.

Zwiazek, J.J., and Blake, T.J. 1989. Effects of preconditioning onsubsequent water relations, stomatal sensitivity and photosyn-thesis in osmotically stressed black spruce. Can. J. Bot.67:2240–2244.



growth, morphology, and gas exchange in white spruce ( picea glauca )...

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