Abstract: Physiological response of interior spruce (Picea glauca (Moench) Voss × Picea engelmannii Parry ex Engelm.) to
drought conditions was compared for somatic seedlings from clones G351, T703, N366, and W460. Seedlings were subjected
to four cycles of progressive soil drought by withholding water. Changes in net photosynthesis (Pn), stomatal conductance
(gwv), and predawn water potentials (ψpd) were measured during and after each drought cycle. Shoot tissue water relations
parameters were measured using pressure–volume analysis at the beginning and end of the fourth drought cycle. When
comparing drought cycle 1 with drought cycle 4, clones G351, N366, and T703 showed significantly reduced Pn, but gwv of all
clones was not affected. Net photosynthesis and gwv decreased with ψpd more rapidly in clone W460 than in the remaining
clones. When rewatered after drought, clone T703 had the most rapid Pn and gwv recovery whereas clone G351 had the
slowest recovery of Pn and gwv. Over four drought cycles, all clones photosynthesized at progressively lower ψpd, but
adjustments in tissue water relations parameters were marginal. These results implied that gas exchange parameters were more
sensitive than shoot tissue water relations parameters in detecting clonal variation in the physiological response of interior
spruce under simulated drought conditions.
Résumé: Nous avons obtenu des semis d’origine somatique à partir des clones G351, T703, N366 et W460 d’épinette de
l’intérieur (Picea glauca (Moench) Voss × Picea engelmannii Parry ex Engelm.), et nous avons comparé leur réponse
physiologique à la sécheresse. Nous avons soumis les semis à quatre cycles progressifs d’assèchement du sol par réduction de
l’arrosage et nous avons mesuré les changements de photosynthèse nette (Pn), de conductance stomatique (gwv) et de
potentiels hydriques pré-aube (ψpd) après chaque cycle. Nous avons aussi mesuré les paramètres des relations hydriques des
tissus de la portion aérienne des plants au début et à la fin du quatrième cycle d’assèchement par l’analyse des courbes
pression–volume. Du cycle 1 au cycle 4, les valeurs de Pn ont été réduites significativement chez les clones G351, N366 et
T703. Les valeurs de gwv sont par contre restées inchangées chez tous les clones. Les valeurs de Pn et de gwv décroissaient plus
rapidement avec une baisse de ψpd chez le clone W460 que chez les autres clones. Après arrosage, la récupération de Pn et de
gwv était la plus rapide chez le clone T703, et la moins rapide chez le clone G351. D’un cycle d’assèchement à l’autre, tous les
clones ont maintenu leur photosynthèse à des valeurs de ψpd progressivement plus faibles, mais l’ajustement des paramètres
des relations hydriques de leurs tissus était marginal. Nous pouvons déduire de ces résultats que, chez l’épinette de l’intérieur,
les paramètres des échanges gazeux sont plus sensibles aux différences interclonales de réponse à la sécheresse que ne le sont
les paramètres des relations hydriques des tissus.
[Traduit par la Rédaction]
Introduction
Physiological processes are phenotypic traits that are deter-mined by genetics and environmental conditions (Kramer1986). Studies on interior spruce (Picea glauca (Moench)Voss × Picea engelmannii Parry ex Engelm.), a major speciesin forestry programs in north-central British Columbia (B.C.)(Coates et al. 1994), have found that changes in physiological
parameters, such as drought and freezing tolerance, and gasexchange characteristics are predictable with variation in thegenetic makeup of the genotypes (Grossnickle et al. 1996; Fanet al. 1997). Interior spruce families that have higher photo-synthetic capacity and water use efficiency are found to bemore productive under both well-watered and water-stressedconditions (Sun et al. 1996). A strong genotype × environmentinteraction in gas exchange and growth was observed in blackspruce (Picea mariana (Mill.) BSP) (Johnsen and Major 1995;Major and Johnsen 1996). Compared with the slower growingfamilies, families that grow faster on drier sites are found tohave higher gas exchange rates (Tan et al. 1992a; Major andJohnsen 1996), higher water use efficiency (Flanagan andJohnsen 1995), osmotic adjustment (Tan et al. 1992b), and lessabscisic acid (ABA) accumulation and electrolyte leakage(Tan and Blake 1993). Norway spruce (Picea abies (L.) Karst.)
Received September 11, 1997. Accepted March 17, 1998.
S. Fan1 and S.C. Grossnickle.Forest Biotechnology Centre,B.C. Research, Inc., 3650 Wesbrook Mall, Vancouver, BCV6S 2L2, Canada.
1 Author to whom all correspondence should be addressed.e-mail: [email protected].
clones differing in growth rates are reportedly different in sus-ceptibilities to ozone stress and soil drought (Karlsson et al.1997). These results suggest that physiological parametersunderlying genetic variation can be useful tools in identifyingand selecting superior genotypes for specific environmentalconditions.
Drought is often a primary environmental condition that hasa great impact on the physiological processes and growth oftrees (Borchert 1991; Kramer 1986), particularly for newlytransplanted seedlings (Burdett 1990; Margolis and Brand1990; Rietveld 1989). Under drought, water stress causesphysiological and biochemical changes that are detrimental,such as photosynthetic inhibition (Grossnickle and Major1994a, 1994b), turgor loss (Hsiao 1973), ABA accumulation(Roberts and Dumbroff 1986; Davies and Zhang 1991), anddisruption of membrane integrity (Tan and Blake 1993; Fanand Blake 1994).
On the other hand, water stress also triggers adaptivemechanisms, such as stomatal closure and osmotic adjustment(Morgan 1984; Tan et al. 1992b; Zwiazek and Blake 1990),elasticity adjustment (Joly and Zaerr 1987; Fan et al. 1994),and total turgor adjustment (Colombo 1987; Grossnickle andRussell 1996), that can increase dehydration avoidance andtolerance. These mechanisms help tree seedlings to survivedrought and to recover after drought is relieved. Tests for geno-typic variation in these mechanisms would shed light on theadaptedness of different genotypes. Thus, physiological mea-surements under simulated stressful conditions may allow oneto predict field performance of different genotypes. In con-junction with field tests, physiological testing may help selectgenotypes that perform well under specific environmentalconditions.
The focus of this research was to examine clonal differ-ences in interior spruce for gas exchange and shoot tissue waterrelations response to progressive soil drought cycles. Theclones were produced for a clonal selection program jointlyconducted by the B.C. Ministry of Forests and B.C. Research,Inc. (Cyr et al. 1994). This study was part of a comprehensiveecophysiological program that is aimed at understandingclonal differences in interior spruce.
Materials and methods
Plant materialsExperiments were conducted on 2 + 0 somatic seedlings of interior
spruce, which is a complex of white spruce (Picea glauca (Moench)Voss), Engelmann spruce (Picea engelmannii Parry ex Engelm.), andtheir natural hybrid (Coates et al. 1994). Seedlings were grown fromsomatic embryos produced from seed of full-sib families throughsomatic embryogenesis tissue culture procedures (Roberts et al. 1991;Webster et al. 1990). The families were created by assortive matingin a tree improvement program for interior spruce conducted by theB.C. Ministry of Forests (Kiss and Yeh 1988). The experimental seed-lings came from clones of six families that had, respectively, a per-centile ranking of 14 (clones G351 and T703), 25 (H103), 50 (M121),and 97% (N366 and W460) for growth rates on a faster-to-slowergrowth scale. These family rankings are based on 10-year heightgrowth in a progeny trial on 170 families being conducted by the B.C.Ministry of Forests, located in the north-central interior of BritishColumbia (53°N, 122°W) (Kiss and Yeh 1988). Each family hadmany clones, and each clone had various numbers of seedlings andnursery growth rates. To maintain seedling uniformity, clones with
the greatest number of seedlings were chosen from the six families.Results were reported for clones G351, N366, T703, and W460 be-cause they covered the range of physiological response of the sixclones.
Somatic seedlings were grown in 415B styroblocks (105 cm3 percavity, 112 cavities per block) (Beaver Plastics Ltd., Edmonton, Alta.)as 1 + 0 stock during the 1993 season under a previously describedgreenhouse culture regime for containerized seedlings at Pelton Re-forestation Ltd., Maple Ridge, B.C. (49°18′N) (Grossnickle et al.1994). In early January 1994, seedlings were lifted and frozen storeduntil mid-March 1994. At this time, they were replanted into 615Bstyroblocks (340 cm3 per cavity, 45 cavities per block) and grownoutdoors as 2 + 0 stock under optimum conditions. This was to ensurethat seedling phenotype would be similar to that of field-grown plantsand that residual tissue culture and greenhouse culture effects wouldbe minimal. In later mid-November 1994, seedlings were moved to anonheated, open-walled greenhouse and allowed to undergo normalfall acclimation processes.
On January 20, 1995, 30 seedlings per clone were selected fromthe population and potted in 2 l cartons (9.5 × 9.5 × 26 cm) filled withfine sand and 6 g of Nutricote slow-release fertilizer 16 (N) – 10(P2O5) – 10 (K2O) (type 180, Sierra Chemical Co., Milpitas, Calif.).Average height (centimetres) and diameter (millimetres) of the se-lected seedlings were, respectively, 18.4 ± 0.2 and 5.54 ± 0.13(G351), 20.2 ± 0.3 and 6.31 ± 0.13 (N366), 20.0 ± 0.4 and 5.57 ± 0.13(T703), and 23.4 ± 0.8 and 6.79 ± 0.16 (W460). The seedlings wererandomly placed on two tables in a controlled-environment growthroom and grown under the following environmental conditions untilMarch 20, 1996, when they had started to set bud: 22/18°C day/nightair temperatures, 40 ± 10% relative humidity, and a 20-h photoperiodof 600 µmol⋅m–2⋅s–1 photosynthetic photon flux density (PPFD) atseedling height, which was provided by two Sylvania 1000-WMetalarc® metal halide lamps (Osram Sylvania Inc., Manchester,N.H.) on each growth table. During this period, seedlings were wellwatered and fertilized once a week with 1.25 g⋅L–1 (250 ppm Nequivalent) 20–8–20 forest seedling fertilizer (Plant Products Co.Ltd., Bramptom, Ont.).
Soil drought treatmentStarting on March 21, 1995, seedlings were subjected to four cy-
cles of soil drought by withholding water. Each of the first threedrought cycles was uninterrupted and the severity of drought wasincreased gradually (Fig. 1). When shoot predawn water potential (ψpd)in seedlings used for gas exchange and pressure–volume analysismeasurements reached a predefined range (approximately –0.6 to–1.0 MPa for cycle 1, –1.2 to –1.6 MPa for cycle 2, and –1.6 to–2.0 MPa for cycle 3), the drought cycle was terminated by rewater-ing for 6 days. During the rewatering period, seedlings were fertilizedtwice as described above.
During the fourth drought cycle, seedlings were droughted andpartially rewatered three times with dilute fertilizer solution (0.25g⋅L–1) (Fig. 1). However, there was no sustained period of optimumsoil moisture following each rewatering as in the first three droughtcycles. When net photosynthesis (Pn) of each selected seedlingreached ≤0, the drought was concluded by thoroughly rewatering theseedling for 14 days, with fertilization (1.25 g⋅L–1) occurring on thefirst, fifth, and 10th days. A group of well-watered seedlings wasgrown along with the droughted seedlings as a control for measure-ment of shoot tissue water relations parameters.
ductance (gwv), and intercellular CO2 concentration (Ci)) were deter-mined using a LI-6200 portable photosynthesis system and a 250-mLleaf chamber (LI-COR, Inc., Lincoln, Nebr.). Measurements weretaken on a recently matured, upper crown lateral branch on eightseedlings per clone. The sample branch was repeatedly measured
throughout the experiment, except in N366 where the sample branchwas changed two or three times because drought caused needle ne-crosis. Environmental parameters in the leaf chamber, such as PPFD,air temperature (Ta), CO2 concentration (Ca), relative humidity (RH),and vapor pressure deficit (VPD), were recorded with each gas ex-change measurement. The pooled results for these environmental pa-rameters across the experimental period (means ± SE) were asfollows: PPFD, 836 ± 4 µmol⋅m–2⋅s–1; Ta, 27.4 ± 0.1°C; Ca, 390.3 ±0.5 µL⋅L–1; RH, 35.9 ± 0.2 %; VPD, 2.41 ± 0.1 kPa. The high tem-perature and low humidity in the leaf chamber were caused by heatfrom the light bulbs on the growth tables, as gas exchange measure-ments were taken directly under the light source.
Gas exchange was measured two or three times per week duringthe first three drought cycles, but more frequently during the fourthdrought cycle, based on the drying rate of each seedling. During therecovery period following the fourth drought cycle, gas exchange wasmeasured on the first, seventh, and 14th days under optimum soilmoisture conditions. Gas exchange measurements were taken begin-ning 3 h after lights were turned on. At the end of the experiment, allsample branches were harvested and oven-dried (at 70°C for 48 h).Needle surface area on each branch was converted from needle weightusing a regression equation previously established for interior spruce(S.C. Grossnickle, unpublished). All gas exchange measurementswere then recalculated based on total needle surface area. Net photo-synthesis and gwv recovery after drought were expressed as a percent-age of their predrought level.
Prior to gas exchange measurement, ψpd was measured on a smalllateral branch tip with a pressure chamber (model 3005, Soil MoistureEquipment Corp., Santa Barbara, Calif.). Measurements of ψpd weretaken at the end of the dark period just before lights were turned on.
Shoot tissue water relations measurementShoot tissue water relations were measured on excised lateral
branches using a pressure chamber and a standard shoot-transpirationpressure–volume method (Grossnickle 1989). Twelve seedlings perclone, six from each of both the droughted and well-watered groups,were measured at the beginning of the fourth drought cycle, and sixdroughted seedlings per clone were measured at the end of the fourthdrought cycle. Osmotic potentials at full turgor (ψπ0) and turgor losspoint (ψπTLP) and relative water content at turgor loss point (RTLP)
were determined for each seedling from the series of fresh weight,water potential, and dry weight measurements (Schulte and Hinckley1985). Total turgor potential (PTotal) was calculated and is defined asthe integral of turgor potential with respect to relative water contentfrom full saturation (100%) to RTLP on the Höfler diagram (Colombo1987; Grossnickle and Russell 1996). This parameter integrates the con-tribution of elastic and osmotic adjustments to turgor maintenance.
Growth measurementAt the beginning and end of the experiment, seedlings of each
clone were measured for height and diameter. Incremental height anddiameter growth accumulated during the experimental period werethen calculated as the difference between these two measurements.
Data analysisSeedlings of the four clones varied in size and drought response.
On each measurement day, individual seedlings were under variousdegrees of water stress. Thus, covariance analysis was used to com-pare clones for Pn and gwv response to declining ψpd, and Pn changeswith respect to gwv during the first and fourth drought cycles. The firstcovariance analysis used ψpd whereas the second analysis used gwv asthe covariant. Covariance analysis was also used to compare clonesfor relative Pn and gwv recovery during the period of optimum soilmoisture after drought. In this analysis, the covariant was the ψpd
measured on the day just before seedlings were rewatered. This wasbased on a finding that Pn and gwv recovery in interior spruce somaticseedlings is determined by the severity of drought before the seedlingswere rewatered (Grossnickle and Fan 1998a).
The relationships between Pn or gwv and ψpd and between Pn andgwv were nonlinear. To meet the linearity requirement, Pn was loga-rithmatically transformed whereas gwv was reciprocally transformedto obtain a linear relationship with the covariant. Data transformationnot only remedies deviations from linearity, but also tends to simulta-neously remove nonnormality and heteroscedasticity to allow a test ofsignificance on nonlinear data (Huitema 1980; Sokal and Rohlf1969). The general statistical model used for covariance analysis onthe transformed data was
Yijk = µ + Ci + Wj + CWij + εijk
where Yijk is the physiological measurement, µ is the clone mean, Ci
Fig. 1. Example showing predawn shoot water potential (ψpd) changes with the increase in days of soil drought during each drought cycle in
interior spruce somatic seedlings (clone T703), as well as the drought and rewatering treatments. Thick arrows indicate the start of rewatering;
thin arrows indicate the start of a drought cycle.
is the effect of the ith clone, Wj is the effect of the jth covariant, CWij
is the interaction of the ith clone with the jth covariant, and εijk is therandom effect. This model was first run using a general linear modelprocedure to test slope homogeneity before an analysis of covariancewas performed using an ANCOVA procedure. In cases of slope het-erogeneity, a Johnson–Neyman analysis (Johnson and Neyman 1936;Huitema 1980) was performed as an alternative to covariance analy-sis. This analysis procedure defines the regions across the covariantscale where significant treatment or clonal effects can be found. The
Bonferroni critical F-values in this analysis were chosen at the 5%level.
Analysis of variance was used to compare drought treatment andclonal effects on photosynthetic acclimation to progressive droughtcycles, shoot tissue water relations parameters, and growth incre-ments, as all of these were single timepoint measurements. All statis-tical analyses used procedures in the Systat® for WindowsTM (version5.0): statistics programs (Wilkinson et al. 1992).
Results
Clonal variation in gas exchange parametersAs shoot ψpd declined in droughted seedlings of interior
spruce clones G351, N366, T703, and W460 (Fig. 1), both gwv
(Fig. 2) and Pn (Fig. 3) decreased. However, the four clonesdiffered in gwv and Pn response to ψpd, which varied withdrought cycles. During the first drought cycle, covarianceanalysis found clonal differences in means, but not in slopes,of gwv (p ≤ 0.027) and Pn (p < 0.0000). Seedlings of T703 andG351 had higher gwv (9.14 and 6.74 mmol⋅m–2⋅s–1 in adjustedleast square means, respectively) and Pn (1.40 and1.13 µmol⋅m–2⋅s–1 in adjusted least square means, respec-tively) than seedlings of W460 and N366, which had a gwv of5.24 and 5.99 mmol⋅m–2⋅s–1 and a Pn of 0.71 and0.98 µmol⋅m–2⋅s–1 (adjusted least square means), respectively.
During the fourth drought cycle, the four clones had differ-ent gwv (p ≤ 0.04) and Pn (p ≤ 0.004) response to ψpd. Theywere separarted into two groups. Seedlings of G351, N366,and T703 were similar in gwv and Pn response to ψpd (Fig. 4).In contrast, seedlings of W460 had a sharper decrease in gwv
and Pn with ψpd. Seedlings of W460 had lower gwv and Pn thanseedlings of G351, T703, and N366 at ψpd lower than –1.34and –1.22 MPa, respectively.
For all four clones, covariance analysis found no droughtcycle effects on gwv (p ≥ 0.2) response to ψpd (Fig. 2), althoughPn was reduced in seedlings of G351 (p ≤ 0.0003), N366(p ≤ 0.0087), and T703 (p ≤ 0.0012) in the fourth drought cycle(Fig. 3). Net photosynthesis for W460 seedlings was unaf-fected (p ≥ 0.24) by repeated drought. The lack of an effect ofprogressive drought cycles on gwv in seedlings of N366 andT703 could have been distorted by the scatter of data pointsfor gwv versus ψpd obtained during the fourth drought cycle(Fig. 2). Virtually all seedlings of N366 suffered severe needlenecrosis under drought. A few big seedlings of T703 alsoshowed needle browning, probably due to repeated severewater stress, since these seedlings often dried up faster thanthe other measured seedlings (Fig. 1). Needle necrosis couldhave reduced gas exchange capability and distorted gas ex-change measurements.
The four clones had no differences (p ≥ 0.25) in Pn responseto gwv during the first drought cycle (Fig. 5). During the fourthdrought cycle, clonal differences (p ≤ 0.047) in Pn were found,with higher Pn in seedlings of G351 than in seedlings of N366,T703, and W460 as gwv decreased. In all four clones, Pn de-creased faster with gwv in the first drought cycle than in thefourth drought cycle (Fig. 5).
The patterns of intercellular to ambient CO2 concentration(Ci/Ca) ratio with the decrease in ψpd were also different be-tween the first and the fourth drought cycles (Fig. 6). The Ci/Ca
ratio either decreased (in T703) or was virtually constant (inremaining clones) in the first drought cycle. In contrast, the
Fig. 2. Changes in stomatal conductance (gwv) in response to
declining shoot predawn water potential (ψpd) in interior spruce
somatic seedlings of clones G351, N366, T703, and W460 for
drought cycles 1 (solid line, open circles) and 4 (broken line, solid
squares). The scattering of data points in clones N366 and T703
resulted from needle damage under soil drought. Lines were drawn
Ci/Ca ratio increased with ψpd in the fourth drought cycle in asimilar fashion for all four clones.
Tolerance of Pn to dehydration increased in seedlings of allfour clones with the progression of drought cycles, as indicatedby the lowering of ψpd at which Pn was equal to zero (Fig. 7).However, there were no clonal differences (p ≥ 0.2) in thisparameter during all four drought cycles.
When seedlings were rewatered after the fourth droughtcycle, the four clones varied (p ≤ 0.01) in the recovery of gwv
and Pn (Fig. 8). Stomatal conductance and Pn in seedlings ofT703 recovered to 100 and 78%, respectively, of their pre-drought level on the sixth day and to 113 and 83% on the14th day after rewatering. In contrast, gwv and Pn in seedlingsof G351 recovered to only 44 and 23%, respectively, of theirpredrought level on the sixth day and to 59 and 28% on the14th day after rewatering. Stomatal conductance in seedlingsof W460 had nearly recovered to its predrought level after6 days of rewatering, but Pn recovered to only 42% of thepredrought level after 14 days of rewatering. Seedlings ofN366 had similar Pn recovery but slower gwv recovery com-pared with seedlings of W460.
Clonal differences in shoot tissue water relationsAt the beginning of the fourth drought cycle, shoot tissue
water relations parameters (ψπ0, ψπTLP, RTLP, and PTotal) ofprogressively droughted seedlings were similar (p ≥ 0.14)among clones (Fig. 9). The only exception was ψπTLP for G351seedlings, which was slightly lower (p ≤ 0.073) than for seed-lings of N366 and W460. There were also few clonal differ-ences in these parameters between the progressively droughtedseedlings and seedlings grown under optimum soil moistureconditions. At the end of the fourth drought cycle, only N366seedlings had a slightly higher (p ≤ 0.05) ψπ0 than seedlings
Fig. 3. Changes in net photosynthesis (Pn) in response to declining
shoot predawn water potential (ψpd) in interior spruce somatic
seedlings of clones G351, N366, T703, and W460 for drought
cycles 1 (solid line, open circles) and 4 (broken line, solid squares).
The scattering of data points in clones N366 and T703 resulted
from needle damage under soil drought. Lines were drawn to
highlight the trend of changes.
Fig. 4. Clonal differences in stomatal conductance (gwv) and net
photosynthesis (Pn) response to declining shoot predawn water
potential (ψpd) measured in the fourth drought cycle. Regressional
lines and equations are shown for transformed data. Small 1/gwv or
negative ln (Pn) values on the graph indicate large real gwv or small
of the other clones. There were no clonal differences (p ≥ 0.3)in ψπTLP, RTLP, and PTotal at the end of the fourth drought cycle.
Clonal variation in growthThere were clonal differences in incremental growth of
height (p < 0.0000) and diameter at root collar (p ≤ 0.0002).During the experimental period, seedlings of G351, N366,T703, and W460 had a height increment of 10.6 ± 0.8, 6.6 ±1.0, 10.4 ± 0.9, and 4.7 ± 1.0 cm, respectively, and a diameterincrement of 0.431 ± 0.020, 0.305 ± 0.024, 0.329 ± 0.022, and0.284 ± 0.028 cm, respectively. Four seedlings of G351 re-
flushed during the recovery period whereas six W460 seed-lings had litter terminal growth during the experimental period.These may have biased the height increment of these clones.
Discussion
Net photosynthesis and gwv of progressively droughted so-matic seedlings of interior spruce clones responded to the de-crease in shoot ψpd in a typical way as previously observed forwhite spruce (Grossnickle and Blake 1987) and interior spruce
Fig. 5. Changes in net photosynthesis (Pn) in response to declining
stomatal conductance (gwv) in interior spruce somatic seedlings of
clones G351, N366, T703, and W460 for drought cycles 1 (solid
line, open circles) and 4 (broken line, solid squares). Lines were
drawn to highlight the trend of changes.
Fig. 6. Changes in the intercellular to ambient CO2 concentration
(Ci/Ca) ratio in response to declining predawn shoot water potential
(ψpd) in interior spruce somatic seedlings of clones G351, N366,
T703, and W460 for drought cycles 1 (solid line, open circles) and
4 (broken line, solid squares). Lines were drawn to highlight the
zygotic seedlings (Fan et al. 1997) and interior spruce somaticseedlings (Grossnickle and Major 1994b). However, the testedinterior spruce clones varied in Pn and gwv response to progres-sively repeated soil drought and rewatering after drought.
Clonal differences in gas exchange response were observedbefore, during, and after drought. Under well-watered condi-tions before drought, seedlings of N366 and W460 tended tohave higher gas exchange rates than seedlings of G351 andT703 as observed in another experiment with these clones(Grossnickle and Fan 1998b). However, under drought, seed-lings of T703 and G351 maintained higher gas exchange ratesthan seedlings of N366 and W460. The decrease of Pn and gwv
with ψpd was also slower in seedlings of T703 and G351 thanin seedlings of N366 and W460 under subsequent drought.Recent studies on Sitka spruce (Picea sitchensis (Bong.) Carr.)× interior spruce introgression hybrids have found that gasexchange rates are genetically determined. Genotypes that aremore drought tolerant (Grossnickle et al. 1996) have highergas exchange rates under drought and recover faster afterdrought (Fan et al. 1997).
Differences in gas exchange response to repeated droughtwith respect to well-watered conditions suggest that these fourclones may have different modes of gas exchange response todrought. Seedlings of T703 were particularly more tolerant torepeated drought than seedlings of the other clones becausethey had the lowest ψpd at which Pn ceased. By comparison,seedlings of N366 were much more susceptible to repeateddrought. Not only did seedlings of this clone have reduced gasexchange capability, but also suffered severe needle damageunder repeated drought. Reduced gas exchange capability it-self may be part of the cause for the needle damage. Low gwv
would have restricted transpiration to reduce transpirationcooling and low Pn may have resulted in the accumulation ofexcessive light energy, which induced temperature increases
and photodamage to the needle tissues. Seedlings of W460seemed to be dehydration avoidant. Their gas exchange capa-bility rapidly decreased with ψpd under drought. Stomatal clo-sure could have reduced Pn, but dehydration-induced injuriesto the photosynthetic apparatus were probably also reduced.This explains why Pn for seedlings of this clone was not sig-nificantly affected by repeated drought.
It has been reported that gas exchange response of spruceto drought and growth in the field are correlated. Black sprucefamilies having higher gas exchange rates and water use effi-ciency on dry sites or under simulated drought are found togrow better than families that have lower gas exchange rates(Tan et al. 1992a; Flanagan and Johnsen 1995; Johnsen andMajor 1995; Major and Johnsen 1996). Interior spruce familieshaving higher Pn and water use efficiency were also found tohave higher productivity whether they were grown under well-watered or water-stressed conditions (Sun et al. 1996). Thereappeared to be a correlation between growth and gas exchangemeasurements among these interior spruce clones. Seedlingsof G351 and T703 had higher gas exchange rates underdrought, and also greater height and diameter growth duringthe experimental period. Long-term field progeny trials con-ducted by the B.C. Ministry of Forests in north-central BritishColumbia have demonstrated that the families to which G351and T703 belong grow faster than the families of N366 andW460 (Kiss and Yeh 1988). Measurements of gas exchangeresponse to drought, in conjunction with field trials, may serveas criteria for selection of interior spruce clones.
Taking gas exchange recovery after drought into considera-tion may further increase the power of the use of gas exchangemeasurements for clonal selection. After four cycles ofdrought, seedlings of T703 had the most rapid recoverywhereas seedlings of G351 had the slowest recovery, withseedlings of N366 and W460 in the middle of these two
Fig. 7. Changes in shoot predawn water potential (ψpn) at which net photosynthesis (Pn) was equal to zero (ψpd Pn=0) with drought cycles in
interior spruce somatic seedlings of clones G351, N366, T703, and W460. The larger than average SE of ψpd Pn=0 in clone G351 during the
first drought cycle was caused by fewer data points (n = 3 versus n = 8).
extremes. During the experimental period, the ψpd at which Pn
ceased was lowered by approximately –1.1 MPa in seedlingsof T703 compared with an adjustment of –0.34, –0.47, and–0.57 MPa in seedlings of clones G351, N366, and W460,respectively. Differences in dehydration tolerance of the pho-tosynthetic apparatus may partly explain clonal variation ingas exchange recovery. Drought recovery is part of the droughtresistance mechanisms inherent in a species. Slow recovery ofgas exchange in seedlings of G351, N366, and W460 com-pared with seedlings of T703 suggests that these clones wereless drought tolerant. On sites prone to frequent drought, theseclones may not perform well.
Despite clonal differences in gas exchange recovery, noneof the four clones had a full Pn recovery 2 weeks after drought.Slower Pn recovery after drought and lower Pn under sub-sequent drought suggested damage to the photosynthetic ap-paratus (Kirschbaum 1988) by repeated drought, possibly theultrastructure of chloroplasts (Palomäki et al. 1994). Couplinggas exchange measurements with chlorophyll fluorescencemeasurements, Eastman and Camm (1995) found that the pho-tosynthetic apparatus of interior spruce seedlings is more sen-sitive to dehydration than other angiosperm species, such asoak (Epron and Dreyer 1993).
The increasing pattern of Ci/Ca ratio with ψpd in the fourthdrought cycle, compared with the flat or decreasing pattern inthe first drought cycle, in seedlings of all four clones was in-dicative of the intolerance of the photosynthetic apparatus of
Fig. 8. Stomatal conductance (gwv) and net photosynthesis (Pn)
recovery after seedlings of clones G351, N366, T703, and W460
were released from the fourth drought cycle. Values are expressed
as the percentage of the predrought level.
Fig. 9. Shoot tissue water relations parameters of interior spruce
somatic seedlings of clones G351, N366, T703, and W460: osmotic
potentials at full turgor (ψπ0) and turgor loss point (ψπTLP), relative
water content at turgor loss point (RTLP), and total turgor (PTotal).
These parameters were measured at the beginning and end of the
fourth drought cycle for seedlings receiving progressive drought cycles
and at the beginning of the fourth drought cycle for control
seedlings grown under optimum soil moisture conditions (n = 6).
interior spruce to dehydration. As gwv was reduced by repeateddrought, lower gwv should have caused the Ci/Ca ratio to de-crease due to increased stomatal limitations to CO2 diffusioninto the needle tissues and the continuing photosynthetic con-sumption of intercellular CO2. Instead, the Ci/Ca ratio contin-ued to rise with ψpd. This implied that Pn itself was inhibitedby repeated drought-induced nonstomatal limitations, explain-ing the lower Pn in the fourth drought cycle than in the firstdrought cycle. Stewart et al. (1995) also reported that nonsto-matal limitations to Pn became increasingly more importantthan stomatal limitations in repeatedly droughted black spruceseedlings. On the other hand, because gwv decreases as Ci in-creases (Farquhar et al. 1987), a higher Ci/Ca ratio could havealso contributed to the lower gwv in the fourth drought cyclethan in the first drought cycle. Because repeated drought gen-erally had damaging effects, clones that were more tolerant torepeated drought, such as T703, should be selected for sites onwhich drought frequents during the growing season.
Drought induces ABA accumulation (Fan and Blake 1994;Roberts and Dumbroff 1986; Tan and Blake 1993), which isknown to cause stomatal closure (Davies and Zhang 1991),disturb membrane integrity by increasing electrolyte leakage(Fan and Blake 1994; Tan and Blake 1993), and inhibit pho-tosynthesis (Raschke and Hedrich 1985; Ward and Bruce1987). ABA level in the needle tissues of repeatedly droughtedblack spruce and jack pine (Pinus banksiana Lamb.) was foundto remain much higher than the predrought level 1 week afterrewatering (Fan 1994). Three days of drought relief was foundinsufficient for ABA level to return to the predrought level inwhite spruce seedlings after a single drought (Roberts andDumbroff 1986). In this experiment, seedlings were relievedfrom drought for only 6 days following each of the first threedrought cycles. ABA level in the needle tissues was probablysustained high enough to limit the photosynthetic process, re-ducing Pn in the later drought cycles and slowing gas exchangerecovery after drought.
Seedlings of all clones continued Pn at lower ψpd undersubsequent drought. The ψpd at which Pn ceased was lower inthe fourth drought cycle (–2.0 ± 0.09 MPa, pooled for allclones) than in the first drought cycle (–1.41 ± 0.09 MPa).Nevertheless, it remains unclear whether this increased capa-bility to photosynthesize at lower ψpd was due to photosyn-thetic acclimation of interior spruce somatic seedlings toprogressive drought cycles because of a so-called “droughtconditioning” effect or due to ontogenetic temporal changesbecause the experiment was conducted on seedlings that hadstarted to set bud. As dormancy progresses, relative stress tol-erance increases in tree seedlings (Lavender 1985) and, spe-cifically, drought tolerance in white spruce (Grossnickle1989). All seedlings of the tested interior spruce clones showedno significant adjustments in shoot water relations parametersas compared with the control seedlings, which had not pre-viously been droughted. This suggested that the repeatedlydroughted seedlings had no significant increase in dehydrationtolerance. Thus, it was highly possible that ontogeneticchanges in drought tolerance were attributable to their in-creased ability to photosynthesize at lower water potential.Stewart et al. (1995) observed a similar phenomenon in repeat-edly droughted black spruce seedlings, which they ascribed toontogenetic effects, not acclimation. Both black spruce(Zwiazek and Blake 1989) and red spruce (Picea rubens Sarg.)
(Seiler and Cazell 1990) showed no photosynthetic acclima-tion when seedlings were exposed to progressive drought cy-cles, although Zine El Abidine et al. (1994) reportedphotosynthetic acclimation for black spruce.
Progressive drought cycles had little effect on shoot tissuewater relations parameters (ψπ0, ψπTLP, RTLP, and PTotal) forseedlings of the four clones. The only exception was that seed-lings receiving progressive drought cycles seemed to haveslightly lower ψπTLP at the end of the fourth drought cycle thancontrol seedlings at the beginning of the fourth drought cycle.Clonal differences in these parameters were marginal. Onlyseedlings of G351 had a slightly lower ψπ0. Other studies havefound accumulation of sugars and amino acids in black (Tanet al. 1992b; Zine El Abidine et al. 1994; Zwiazek and Blake1990) and white spruce (Cyr et al. 1990) and synthesis of stressresponsive proteins in jack pine seedlings (Mayne et al. 1994)in response to progressive drought cycles. These hydrophilicagents are suggested to have contributed to osmotic adjustment(Morgan 1984). In this study, these osmolytes were not mea-sured on the interior spruce clones, although one can speculatethat they did not accumulate at the magnitude reported in theother studies. Lack of differences in osmotic adjustment wouldpartly explain the similarities in PTotal among the clones. In thisexperiment, seedlings were grown in sand in small pots. Re-strictions on root growth, nutrient deficiency, and rapid devel-opment of water stress were possible explanations for thenonsignificant ajustment in tissue water relations parametersin the seedlings of these clones.
In conclusion, there were significant clonal differences ininterior spruce in gas exchange response to progressively re-peated soil drought. Clone T703 seemed to be the most dehy-dration-tolerant clone that was tested. In contrast, clone W460appeared to be more dehydration avoidant than the other testedclones. Coupled with field clonal testing, gas exchange mea-surements under simulated conditions could be used for selec-tion of interior spruce clones. However, measurements of Pn
tolerance to dehydration and adjustments of shoot tissue waterrelations parameters were much less variable in these clones,due possibly to the rapid development of soil drought in thisexperiment. Slow stress may allow time for seedlings to de-velop these drought responsive mechanisms. This approachneeds to be tested. The test clones came from families thatwere selected from a narrow selection unit within a singlebiogeoclimatic subzone in the Prince George region of north-central British Columbia. Although the results appear promis-ing, research needs to be extended across a larger array ofclones from more families to assess if these physiological testsof drought tolerance are broadly applicable and useful.
Acknowledgments
Financial support for this program was provided by grantsfrom National Research Council of Canada and the B.C. Min-istry of Forests. This work was conducted while Shihe Fan wasa postdoctoral fellow supported by the Natural Sciences andEngineering Research Council of Canada.
References
Borchert, R. 1991. Growth periodicity and dormancy. In Physiologyof trees. Edited by A.S. Raghavendra. John Wiley & Sons, Inc.,Toronto, Ont. pp. 221–245.
Burdett, A.N. 1990. Physiological processes in plantation estab-lishment and the development of specification for forest plantingstock. Can. J. For. Res. 20: 415–427.
Coates, K.D., Haeussler, S., Lindeburgh, S., Pojar, R., and Stock, A.J.1994. Ecology and silviculture of interior spruce in British Colum-bia. Canada – British Columbia Partnership Agreement on ForestResource Development: FRDA Report 220. Canadian ForestService and B.C. Ministry of Forests.
Colombo, S.J. 1987. Changes in osmotic potential, cell elasticity, andturgor relationships of second-year black spruce container seed-lings. Can. J. For. Res. 7: 365–369.
Cyr, D.R., Buxton, G.F., Webb, D.P., and Dumbroff, E.B. 1990. Ac-cumulation of free amino acids in the shoots and roots of threenorthern conifers during drought. Tree Physiol. 6: 293–303.
Cyr, D.R, Lazaroff, W., Grimes, S., Quan, G., Bethune, T., Dunstan,D., and Roberts, D. 1994. Cryopreservation of interior spruce(Picea glauca engelmanni complex) embryogenic cultures. PlantCell Rep. 13: 574–577.
Davies, W.J., and Zhang, J.H. 1991. Root signals and the regulationof growth and development of plants in drying soil. Annu. Rev.Plant Physiol. Plant Mol. Biol. 42: 55–76.
Eastman, P.A.K., and Camm, E.L. 1995. Regulation of photosynthe-sis in interior spruce during water stress: changes in gas exchangeand chlorophyll fluorescence. Tree Physiol. 15: 229–235.
Epron, D., and Dreyer, E. 1993. Photosynthesis of oak leaves underwater stress: maintenance of high photochemical efficiency ofphotosystem II and occurrence of non uniform CO2 assimilation.Tree Physiol. 13: 107–117.
Fan, S. 1994. Drought tolerance of tree species from different ecologi-cal zones. Ph.D. thesis, University of Toronto, Toronto, Ont.
Fan, S., and Blake, T.J. 1994. Abscisic acid induced electrolyte leak-age in woody species with contrasting ecological requirements.Physiol. Plant. 90: 414–419.
Fan, S., Blake, T.J., and Blumwald, E. 1994. The relative contributionof elastic and osmotic adjustments to turgor maintenance ofwoody species. Physiol. Plant. 90: 408–413.
Fan, S., Grossnickle, S.C., and Sutton, B.C.S. 1997. Relationshipsbetween gas exchange adaptation of Sitka × interior spruce geno-types and ribosomal DNA markers. Tree Physiol. 17: 115–123.
Farquhar, G.D., Hubick, K.T., Terashima, I., Condon, A.G., and Re-chards, R.A. 1987. Genetic variation in the relationship betweenphotosynthetic CO2 assimilation and stomatal conductance towater loss. In Progress in photosynthesis. IV. Edited by J. Biggins.Martinus Nijhoff, Dordrecht, The Netherlands. pp. 209–212.
Flanagan, L.B., and Johnsen, K.H. 1995. Genetic variation in carbonisotope discrimination and its relationship to growth under fieldconditions in full-sib families of Picea mariana. Can. J. For. Res.25: 39–47.
Grossnickle, S.C. 1989. Shoot phenology and water relations of Piceaglauca. Can. J. For. Res. 19: 1287–1290.
Grossnickle, S.C., and Blake, T.J. 1987. Water relations and morpho-logical development of bare-root jack pine and white spruce seed-lings: seedling establishment on a boreal cut-over site. For. Ecol.Manage. 18: 299–318.
Grossnickle, S.C., and Fan, S. 1998a. Genetic variation in response todrought of interior spruce (Picea glauca (Moench) Voss ×P. engelmannii Parry ex Engelm.). Scand. J. For. Res. 13. In press.
Grossnickle, S.C., and Fan, S. 1998b. Genetic variation in summergas exchange patterns of interior spruce (Picea glauca (Moench)Voss × Picea engelmanni Parry ex Engelm.). Can. J. For. Res. 28:831–840.
Grossnickle, S.C., and Major, J.E. 1994a. Interior spruce seedlingscompared with emblings produced from somatic embryogenesis.II. Stock quality assessment prior to field planting. Can. J. For.Res. 24: 1385–1396.
Grossnickle, S.C., and Major, J.E. 1994b. Interior spruce seedlingscompared with emblings produced from somatic embryogenesis.
III. Physiological response and morphological development on areforestation site. Can. J. For. Res. 24: 1397–1407.
Grossnickle, S.C., and Russell, J.H. 1996. Changes in shoot waterrelations parameters of yellow-cedar (Chamaecyparis nootkaten-sis) in response to environmental conditions. Can. J. Bot. 74:31–39.
Grossnickle, S.C., Major, J.E., and Folk, R.S. 1994. Interior spruceseedlings compared with emblings produced from somatic em-bryogenesis. I. Nursery development, fall acclimation, and over-winter storage. Can. J. For. Res. 24: 1376–1384.
Grossnickle, S.C., Sutton, B.C.S., Folk, R.S., and Gawley, J.R. 1996.Relationship between nuclear DNA markers and physiological pa-rameters for Sitka × interior spruce populations. Tree Physiol. 16:547–556.
Hsiao, T.C. 1973. Plant responses to water stress. Annu. Rev. PlantPhysiol. 24: 519–570.
Huitema, B.E. 1980. The analysis of covariance and alternatives. JohnWiley & Sons, Inc., Toronto, Ont. pp 175–184, 270–297.
Johnsen, K.H., and Major, J.E. 1995. Gas exchange of 20-year-oldblack spruce families displaying a genotype × environment inter-action in growth rate. Can. J. For. Res. 25: 430–439.
Johnson, P.O., and Neyman, J. 1936. Tests of certain linear hypothe-ses and their application to some educational problems. Stat. Res.Mem. 1: 57–93.
Joly, R.J., and Zaerr, J.B. 1987. alteration of cell-wall water contentand elasticity in Douglas-fir during periods of water deficits. PlantPhysiol. 83: 418–422.
Karlsson, P.E., Medin, E.L., Wallin, G., Sellden, G., and Sharby, L.1997. Effects of ozone and drought stress on the physiology andgrowth of two clones of Norway spruce (Picea abies). New Phy-tol. 136: 265–275.
Kirschbaum, M.U.F. 1988. Recovery of photosynthesis from waterstress in Eucalyptus paucilora — a process in two stages. PlantCell Environ. 11: 685–694.
Kiss, G.K., and Yeh, F.C. 1988. Heritability estimates for height foryoung interior spruce in British Columbia. Can. J. For. Res. 18:158–162.
Kramer, P.J. 1986. The role of physiology in forestry. Tree Physiol.2: 1–16.
Lavender, D.P. 1985. Bud dormancy. In Evaluating seedling quality:principles, procedures, and predictive abilities of major tests. Pro-ceedings of the Workshop held October 16–18, 1984. Edited byM.L. Duryea. Oregon State University, Corvallis, Oreg.
Major, J.E., and Johnsen, K.H. 1996. Family variation in photosyn-thesis of 22-year-old black spruce: a test of two models of physi-ological response to water stress. Can. J. For. Res. 26: 1922–1933.
Margolis, H.A., and Brand, D.G. 1990. An ecophysiological basis forunderstanding plantation establishment. Can. J. For. Res. 20:375–390.
Mayne, M.B., Subramanian, M., Blake, T.J., Coleman, J.R., andBlumwald, E. 1994. Changes in protein synthesis during droughtconditioning in roots of jack pine seedlings (Pinus banksianaLamb.). Tree Physiol. 14: 509–519.
Morgan, J.M. 1984. Osmoregulation and water stress in higher plants.Annu. Rev. Plant Physiol. 35: 299–319.
Palomäki, V., Holopainen, J.K., and Holopainen, T. 1994. Effects ofdrought and waterlogging on ultrastructure of Scots pine and Nor-way spruce needles. Trees (Berl.), 9: 98–105.
Raschke, K., and Hedrich, R. 1985. Simultaneous and independenteffect of abscisic acid on stomata and the photosynthetic apparatusin whole leaves. Planta (Heidelb.), 163: 105–118.
Rietveld, W.J. 1989. Transplanting stress in bareroot conifer seed-lings: its development and progression to establishment. North. J.Appl. For. 6: 99–107.
Roberts, D.R., and Dumbroff, E.B. 1986. Relationships betweendrought resistance, transpiration rates, and abscisic acid levels inthree northern conifers. Tree Physiol. 1: 161–167.
Roberts, D.R., Webster, F.B., Flinn, B.S., Lazaroff, W.R., McInnis,S.M., and Sutton, B.C.S. 1991. Application of somatic embryo-genesis to clonal propagation of interior spruce. In Woody plantbiotechnology. Edited by M.R. Ahuja. Plenum Press, New York.pp. 157–169.
Schulte, P.J., and Hinckley, T.M. 1985. A comparison of pressure–volume curve data analysis techniques. J. Exp. Bot. 36:1590–1602.
Seiler, J.R., and Cazell, B.H. 1990. Influence of water stress on thephysiology and growth of red spruce seedlings. Tree Physiol. 6:69–78.
Sokal, R.R., and Rohlf, F.J. 1969. Biometry: the principles and prac-tice of statistics in biological research. W.H. Freeman, San Fran-cisco, Calif.
Stewart, J.D., Zine El Abidine, A., and Bernier, P.Y. 1995. Stomataland mesophyll limitations of photosynthesis in black spruce seed-lings during multiple cycles of drought. Tree Physiol. 15: 57–64.
Sun, Z.J., Livingston, N.J., Guy, R.D., and Ethier, G.J. 1996. Stablecarbon isotopes as indicators of increased water use efficiency andproductivity in white spruce (Picea glauca (Moench) Voss) seed-lings. Plant Cell Environ. 19: 887–894.
Tan, W., and Blake, T.J. 1993. Drought tolerance, abscisic acid andelectrolyte leakage in fast- and slow-growing black spruce (Piceamariana) progenies. Physiol. Plant. 89: 817–823.
Tan, W., Blake, T.J., and Boyle, T.J.B. 1992a. Drought tolerance infaster- and slower-growing black spruce (Picea mariana) proge-
nies: I. Stomatal and gas exchange responses to osmotic stress.Physiol. Plant. 85: 639–644.
Tan, W., Blake, T.J., and Boyle, T.J.B. 1992b. Drought tolerance infaster- and slower-growing black spruce (Picea mariana) proge-nies: II. Osmotic adjustment and changes of soluble carbohydratesand amino acids under osmotic stress. Physiol. Plant. 85: 645–651.
Ward, D.A., and Bruce, J.A. 1987. Abscisic acid simultaneously de-creases carboxylation efficiency and quantum yield in attachedsoybean leaves. J. Exp. Bot. 38: 1182–1192.
Webster, F.B., Roberts, D.R., McInnis, S.M., and Sutton, B.C.S.1990. Propagation of interior spruce by somatic embryogenesis.Can. J. For. Res. 20: 1759–1765.
Wilkinson, L., Hill, M., Welna, J.P., and Birkenbeuel, G.K. 1992.Systat® for WindowsTM (version 5.0): statistics. SPSS, Inc., Chi-cago, Ill.
Zine El Abidine, A., Bernier, P.Y., Stewart, J.D., and Plamondon,A.P. 1994. Water stress preconditioning of black spruce seedlingsfrom lowland and upland sites. Can. J. Bot. 72: 1511–1518.
Zwiazek, J.J., and Blake, T.J. 1989. Effects of preconditioning onsubsequent water relations, stomatal sensitivity and photosynthe-sis in osmotically stressed black spruce. Can. J. Bot. 67:2240–2244.
Zwiazek, J.J., and Blake, T.J. 1990. Effects of preconditioning oncarbohydrate and amino acid composition of osmotically stressedblack spruce (Picea mariana) cuttings. Can. J. For. Res. 20:108–112.
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