Directory UMM :Data Elmu:jurnal:T:Tree Physiology:Vol15.1995:
Tree Physiology 15, 57--64
© 1994 Heron Publishing----Victoria, Canada
Stomatal and mesophyll limitations of photosynthesis in black spruce
seedlings during multiple cycles of drought
J. D. STEWART,1,2 A. ZINE EL ABIDINE2 and P. Y. BERNIER1
1
Forestry Canada, Quebec Region, C.P. 3800, Sainte-Foy, Quebec G1V 4C7, Canada
2
Centre de Recherche en Biologie Forestière, Faculté de Foresterie et de Géomatique, Université Laval, Sainte-Foy, Québec G1K 7P4, Canada
Received June 7, 1993
Summary Container-grown black spruce (Picea mariana
(Mill.) B.S.P.) seedlings were planted in trays containing a sand
and peat mixture, and placed in a climate-controlled greenhouse. One group of seedlings was kept well-watered, and
another group was subjected to three cycles of drought. Gas
exchange analysis showed that mesophyll photosynthetic function was largely unimpaired by drought. In contrast, stomatal
conductance was sensitive to drought, although it became less
sensitive with each drought cycle. Both stomatal and mesophyll conductances increased with time in control and droughtstressed seedlings, but mesophyll conductance increased with
time more rapidly than did stomatal conductance. Limitation
of photosynthetic rate was dominated by the mesophyll. In
control seedlings, relative stomatal limitation increased from 6
to 16% by the end of the experiment. In drought-stressed
seedlings, relative stomatal limitation of photosynthesis
reached 40% during the first drought, but decreased to near
control values immediately after rewatering. Because the third,
most severe drought had only a minor effect on stomatal
conductance, relative stomatal limitation of photosynthesis
was similar to that in control seedlings by the end of the
experiment. Inhibition of ontogenetic change during drought
stress may be responsible for the apparent acclimation of
mesophyll photosynthetic processes. We conclude that it would
be more effective to select for high photosynthetic capacity
than for reduced stomatal sensitivity when breeding for increased drought resistance in black spruce seedlings.
Keywords: acclimation, container stock, drought resistance,
mesophyll conductance, Picea mariana, stomatal limitation,
stomatal sensitivity.
of conifer seedlings, any physiological or morphological adjustment that ameliorates the growth reduction due to water
stress will improve their rate of establishment.
The growth of a plant depends on a series of physical and
biochemical processes that transfer CO2 from the surrounding
air into organic molecules, and, for this reason, much work on
acclimation to water stress in trees has focused on the role of
stomata in limiting gas exchange in response to stress (e.g.,
Unterscheutz et al. 1974, Seiler 1985, van den Driessche
1991). Because mesophyll processes involved in the transfer of
CO2 into the chloroplasts and CO2 fixation may also be limiting during drought stress, there have been several studies of
photosynthetic as well as stomatal acclimation to water stress
in trees (e.g., Seiler and Johnson 1985, 1988, Walters and
Reich 1989). However, few studies have examined the relative
contributions of stomatal and nonstomatal acclimation to net
photosynthetic acclimation to drought in trees (Epron and
Dreyer 1990, Seiler and Cazell 1990) and nonwoody plants
(Hutmacher and Krieg 1983, Matthews and Boyer 1984, Cornic et al. 1987).
To identify the traits that contribute most to drought acclimation in black spruce seedlings, we subjected seedlings to
three cycles of drought and measured their photosynthetic
responses to CO2 during the drought and recovery periods. The
objectives of this study were (a) to test for acclimation to
drought stress in net photosynthesis of black spruce seedlings
over three drought cycles, (b) to determine the relative stomatal and nonstomatal contributions to the drought response,
and (c) to identify the mesophyll processes, if any, involved in
acclimation.
Materials and methods
Introduction
Young conifer seedlings are subject to a variety of environmental stresses when they are planted on clear-cut sites. Of
these stresses, drought is perhaps the most prevalent (Burdett
1990). Even in the boreal forest, a decrease in water content of
the surface horizons of the soil can subject shallow-rooted,
newly planted seedlings to severe water stress. Seedlings that
can acclimate to the first drought may be better able to survive
subsequent droughts. Because water stress inhibits the growth
Seedling growing conditions
Black spruce seeds from a lowland Quebec population were
planted in nursery containers (Rigipot, IPL, St-Damien, Quebec) with 50 ml cavities filled with a 3/1 (v/v) peat/vermiculite
medium. Seedlings were raised under standard conditions in a
commercial greenhouse for one growing season, hardened
outside and then placed in black plastic bags in a coldroom at
2 °C for 3 months.
58
STEWART, ZINE EL ABIDINE AND BERNIER
In January, the seedlings were moved to a greenhouse and
transplanted from the nursery containers to 15 × 60 cm trays,
each containing 10 seedlings. The trays were filled to a depth
of 10 cm with a 3/1 (v/v) fine sand/peat mixture. Trays were
watered daily by an automatic overhead watering boom
equipped with fine spray nozzles. Seedlings were fertilized
using the watering system twice per week, with each tray
receiving approximately 47 mg N,P,K (20-20-20) and 1.5 mg
MgSO4. Greenhouse day/night temperatures were 19--24/11-20 °C, and day/night relative humidity was 20--90/65--100%;
both varied with external weather conditions. The seedlings
were maintained in a 16-h photoperiod using high pressure
sodium vapor lamps (Lumalux GTE, Sylvania Canada Ltd.)
that provided 120 µmol m −2 s −1 PAR at seedling level. Tray
positions on the bench were randomized twice a week.
Bud burst occurred in early February, and the seedlings were
held under constant conditions until shoot elongation was
completed. At the beginning of the treatment period, seedlings
averaged 14.0 cm in height and 1.7 cm in diameter.
Cyclic drought and recovery treatment
Beginning March 11, the cyclic drought and recovery treatment was initiated by withholding water from the seedlings.
Control seedlings continued to be watered daily by hand.
Drought was allowed to progress until turgor loss (wilting)
became visible in the current shoots of some seedlings, whereupon the drought was terminated. Three such drought episodes
lasting 10, 10 and 8 days, respectively, were completed. Each
drought period was followed by a 7-day recovery period,
during which all seedlings were watered daily and fertilized as
described above. Each pair of drought and recovery periods
comprised a drought treatment cycle, and the three cycles
occupied 49 days. Soil water potential (Ψsoil) was measured
with calibrated gypsum blocks (Soilmoisture Equipment Corporation, Santa Barbara, CA), and predawn xylem water potential (Ψx) was measured with a pressure chamber (PMS
Instruments, Corvallis, OR) in neighboring nonexperimental
trays. The severity of the droughts as measured by soil and
predawn xylem water potentials is shown in Table 1 (see also
Zine El Abidine et al. 1995).
At intervals during the drought and recovery cycles, one
seedling from each of four trays was selected at random for gas
exchange measurements. Each seedling was excavated and
placed in a Ray Leach ‘‘Conetainer’’ (Stuewe and Sons, Corvallis, OR) in a controlled environment chamber. On each
measurement day, all four seedlings were either from the
control group or from the treatment group.
The response of net photosynthesis (Anet) to intercellular
CO2 concentration (ci), i.e., the A/ci relationship, was measured
by the nonsteady state method (Davis et al. 1987, McDermit et
al. 1989) in a closed-circuit gas analysis system (LI-6200,
Li-Cor Inc., Lincoln, NB). To standardize the measurement
process, the seedlings were placed in a controlled environment
chamber and kept in the dark in a loosely closed box until
needed. The concentration of CO2 in the chamber was elevated
as a result of respiration by the observer and was crudely
regulated to between 600 and 700 µmol mol −1. Water-cooled
high pressure sodium and mercury lamps were suspended
above the cuvette and provided 1000 µmol m 2 s −1 PAR at the
level of the cuvette. For additional heat dissipation, a transparent acrylic tray filled with water was placed between the lamp
and the cuvette. A fan circulated ambient air around the cuvette
to increase convective cooling. Each seedling was allowed to
adjust to the measurement light conditions for 30 min before
being placed in the 4-l cuvette. Once the seedling was sealed
in the gas exchange cuvette, the relative humidity was raised
to 85% by pumping ambient air through a gasing wash bottle,
filled with water at room temperature, into the gas exchange
system in open mode. The high humidity represented the upper
calibration limit of the humidity sensor (personal communication, J.M. Welles, Li-Cor Inc., Lincoln, NB) and was used to
avoid stomatal closure in response to high VPD in the waterstressed seedlings (Li-Cor 1991), which would have prevented
the gas exchange analysis and is known to provoke heterogeneous stomatal closure in some species. After closure, the
system was equilibrated under the measurement conditions for
20 to 30 min before measurements were taken.
Gas exchange measurements were taken continuously as the
seedling depleted the CO2 in the closed system. Observations
of CO2 and water fluxes were averaged for every 10 µmol
mol −1 change in CO2 concentration. Under severe water stress,
some seedlings had completely closed stomata, and the gas
exchange was too slow to be measured. These seedlings were
not included in the analysis.
Gas exchange parameters calculated were Anet, ci, CO2 compensation point (Γ), transpiration rate (E) and stomatal conductance to water vapor (gs). The program of the LI-6200 was
Table 1. Midday soil water potentials (Ψsoil) and predawn xylem water potentials (Ψx) measured at the end of each drought period before
rewatering. Means ± SE and number of observations are presented. All four soil water potentials measured at the end of the third drought were at
or beyond the range of the gypsum resistance blocks used.
Variable
Drought
Midday soil (MPa)
Treatment
Predawn
Control
Treatment
x (MPa)
1
2
3
−1.18 ± 0.520 (3)
−0.41 ± 0.139 (4)
> −2.00 * (4)
−0.40 ± 0.020 (4)
−1.17 ± 0.091 (15)
−0.75 ± 0.052 (16)
−0.98 ± 0.153 (16)
−0.59 ± 0.034 (16)
−2.62 ± 0.162 (16)
PHOTOSYNTHETIC LIMITATION BY DROUGHT
altered to account for the leak rate of the system as described
in Li-Cor (1991). When the CO2 depletion rate slowed in the
lower part of the curve, CO2 was removed by opening the CO2
scrubbing circuit of the LI-6200 for 45 s, followed by a 5-min
equilibration time before logging of observations resumed.
The average change in CO2 concentration per unit time during
the scrub cycle was similar to that during the continuous upper
portion of the curve. Ambient CO2 concentration used for leak
rate corrections was measured immediately after finishing a
curve. The needles were dried for 48 h in a forced air oven at
70 °C, and dry weights of new and old needles were determined on cooled samples.
Curve-fitting and parameter calculations
Various nonlinear functions were fitted to the CO2 response
(A/ci) data. Two three-parameter functions, the monomolecular
(or Mitscherlich) function and the rectangular hyperbolic function, did not produce satisfactory fits. The four-parameter
nonrectangular hyperbola suggested by Jones (1983) gave the
best fit to the data:
Anet =
Amax (ci − Γ − Anet Ri)
.
Km + ci − Γ − Anet Ri
(1)
The fitted parameters (Km, Amax, Γ and Ri) of the hyperbolic
function correspond to physiological properties of the photosynthetic system. The symbol Amax is the CO2 saturated rate of
net photosynthesis and represents the maximum rate of RuBP
regeneration by the Calvin cycle (Farquhar et al. 1980, von
Caemmerer and Farquhar 1981). The symbol Γ is the CO2
compensation point, which is primarily determined by photorespiration rates. The symbol Km is a constant for Rubisco
affinity for CO2, but it may also be influenced by the light
reactions (Jones 1983). The symbol Ri is a measure of the
resistance to liquid phase transport of CO2 within the mesophyll, but may also be determined by the activity of the carboxylase which may be acting well below its maximum rate in
the lower, near-linear portion of the response curve (Jones
1983). The carboxylation efficiency of Rubisco associated
with changes in concentration or activity (Vrubisco) was calculated from the first derivative of the function, evaluated at A =
0, and is closely related to 1/Ri.
Stomatal and mesophyll conductances to CO2 for an atmospheric concentration of 350 µmol mol −1 were obtained from
the response curve data following the procedure described by
Jones (1985). Stomatal conductance (gs) defined the slope of
the supply curve connecting ca with ci on the demand curve.
Mesophyll conductance (gm) was calculated as the slope of the
demand (A/ci) curve at its intersection with the supply curve.
Stomatal limitation of photosynthesis (Ls) was calculated as in
Jones (1985, Equation 17):
Ls =
1/gs
.
1/gs + 1/gm
(2)
59
Statistical analysis
For the control seedlings, the calculated variables were tested
for linear and quadratic responses over the four control measurement dates. Analyses of variance and a priori contrasts
were performed to assess (a) the differences in response among
the three drought cycles, and (b) the effect of the drought and
recovery treatment versus control conditions. Before the
analysis of variance, data variables were transformed as necessary to uphold the assumptions of normality and heteroscedasticity for the analysis of variance. No transformation was
required for Amax, Vrubisco and gs. All analyses were performed
using the General Linear Models procedure of SAS. Effects
were considered statistically significant when the probability
of a Type I error was 0.05 or less.
Results
Conductance to CO2 and photosynthetic limitation
Stomatal conductance of control seedlings increased with
seedling age, though only during Cycle 3 (Figure 1). Seedlings
recovering from drought showed a similar trend, but stomatal
conductance was generally lower in recovering seedlings than
in control seedlings, especially in Cycles 1 and 3 (Cycle 2
Figure 1. Panel (a) shows the responses of stomatal conductance
(circles) and mesophyll conductance (triangles) of black spruce seedlings to three cycles of drought and recovery. Panel (b) shows the
relative stomatal limitation of photosynthesis (Ls). Open symbols
represent control seedlings and closed symbols represent treatment
seedlings. Each point and bar represent the mean and SE of four
seedlings, except for the point marked by an asterisk, which represents
only one seedling. The timing of the droughts is indicated by the solid
horizontal bars at the bottom of the figure.
60
STEWART, ZINE EL ABIDINE AND BERNIER
produced only a mild stress; Table 1). Drought caused a large
decrease in stomatal conductance, but recovery following rewatering was rapid. The drought-induced reduction in stomatal
conductance was least in Cycle 3, even though this was the
most severe drought.
Mesophyll conductance increased linearly with age in control (Table 2) and drought-stressed seedlings (Figure 1), and
there was no significant difference between control and
drought-stressed seedlings (Table 3).
In control seedlings, the relative stomatal limitation of photosynthesis increased linearly with age (Table 2), rising from
6% at the beginning of the experiment to about 16% at the end
(Figure 1). Stomatal limitation increased to about 40% during
the period of maximum drought stress in Cycle 1, but the effect
was much less during the drought periods in Cycles 2 and 3
(Figure 1). Relative stomatal limitation quickly declined to
near control values on rewatering, and there were no significant differences between recovering and control seedlings in
Cycles 2 and 3 (Table 3). Overall, the rate of photosynthesis
was less affected by stomatal conductance than by mesophyll
conductance.
Gas exchange parameters
Most of the gas exchange parameters measured in control
seedlings increased with time (Table 2 and Figure 2), whereas
the effect of drought varied among the gas exchange parameters and drought cycles.
Gas exchange parameters of both control and recovering
seedlings showed increases mostly in Cycle 3 (Table 3); however, Γ also increased from Cycle 1 to Cycle 2. There was a
significant difference in Γ between control and recovering
seedlings late in Cycles 1 and 2 (Table 2 and Figure 2). By
Cycle 3, Γ was lower in recovering seedlings than in control
seedlings. Both control and recovering seedlings exhibited
similar Amax. Drought only affected Amax during Cycles 1 and 2
(Figure 2 and Table 3). The value of Km decreased with
drought, but tended to recover to values greater than those of
control seedlings on rewatering, but this effect was only statistically significant in Cycle 2 (Table 2 and Figure 2). There was
a significant difference in Vrubisco between control and recovering seedlings late in Cycle 2 (Table 2 and Figure 2). Drought
only affected Vrubisco during Cycle 2.
Discussion
Table 2. Regression analysis of the linear response of the measured
variables in control seedlings with respect to time. The parameter
estimates for intercept and slope, and the t-test probability that slope
is not equal to zero are presented.
Variable
Intercept
Slope
Probability
gs
gm
Ls
Amax
Γ
Km
Vrubisco
1.64
0.035
0.048
59.5
−0.089
4.45
0.261
0.0114
0.0049
0.0025
1.47
0.934
0.248
0.0046
0.070
0.001
0.002
0.002
0.001
0.002
0.001
Ontogenetic changes
As seedlings go through a series of drought cycles, they respond not only to water stress, but also to increasing age and
ontogenetic state. The A/ci curves of control seedlings showed
changes in photosynthetic properties over time. The values of
Amax, Vrubisco and Km increased with age (Figure 2), resulting in
an increase in mesophyll conductance with age (Figure 1). All
of these processes changed in concert, suggesting that this
ontogenetic response is a reflection of the increasing photosynthetic competence of the needles as they mature (Ludlow and
Wilson 1971, Field 1987).
The apparent trend in Γ may not be real, because values
Table 3. Statistical summary for gas exchange and photosynthetic parameters for control seedlings and treatment seedlings at the end of the
recovery periods following each drought. Probability values from a two-way ANOVA with contrasts are presented.
Effect
df
gs
gm
Cycle
Treatment
Cycle × treatment
2
1
2
0.002
0.004
0.441
0.001
0.498
0.673
Contrasts----Controls
Cycle 3 versus 1 + 2
Cycle 1 versus 2
2
1
0.012
0.423
Contrasts----Recovering
Cycle 3 versus 1 + 2
Cycle 1 versus 2
2
1
Amax
G
Km
Vrubisco
0.018
0.080
0.383
0.000
0.690
0.934
0.000
0.015
0.003
0.001
0.010
0.294
0.000
0.042
0.508
0.006
0.122
0.027
0.066
0.006
0.428
0.000
0.000
0.029
0.499
0.001
0.072
0.024
0.336
0.033
0.647
0.222
0.995
0.003
0.680
0.000
0.000
0.003
0.008
0.004
0.818
Contrasts----Controls versus recovering
Cycle 1
1
0.015
Cycle 2
1
0.382
Cycle 3
1
0.046
0.273
0.979
0.926
0.043
0.829
0.428
0.891
0.602
0.974
0.495
0.597
0.000
0.595
0.011
0.129
0.674
0.050
0.220
Error mean square
0.0268
0.304
556
44964
0.0221
0.00419
18
0.0720
Ls
PHOTOSYNTHETIC LIMITATION BY DROUGHT
61
Figure 2. The response of photosynthetic parameters of black
spruce seedlings to three cycles of
drought and recovery. Panels: (a)
CO2 compensation point, (b) affinity constant for carbon fixation,
(c) CO2 saturated maximum net
photosynthetic rate, and (d) maximum rate of carboxylation of
RuBP. The units for Vrubisco are
nmol CO2 g − 1 s − 1 per µmol
mol − 1 CO2. Open symbols represent control seedlings and closed
symbols represent treatment seedlings.
below 10 µmol mol −1 are not likely to occur in a C3 plant
(normal range is 30 to 70 µmol mol −1). The data points at the
bottom of the curve, which have the greatest influence on the
value of Γ, have a lower precision than those at the top of the
curve, due to the very low rates of gas exchange at low ca.
Unlike the other gas exchange parameters, the calculated values of Γ were sensitive to the form of equation used to fit the
data. The monomolecular function gave more realistic and
consistent values of Γ than the nonrectangular hyberbolic
function, but the overall fit was not as good, especially for the
upper portion of the curve.
Stomatal conductance increased with age. Relative stomatal
limitation of photosynthesis also increased with age, because
mesophyll conductance increased faster than stomatal conductance (Table 2 and Figure 2). Although both gs and gm increase
with age in most plants, the relative rates of increase vary
among species (e.g., Ludlow and Wilson 1971, Field 1987,
Mebrahtu and Hanover 1991). Consequently, the change in
relative stomatal limitation of photosynthesis can be positive,
negative or constant, depending on the species.
Acclimation
Acclimation in drought-stressed seedlings is manifested as
either a change in the degree of response to drought, or a
persistent difference between recovering seedlings and the
controls. Although the values of photosynthetic variables in
treated and control seedlings followed similar trends with
time, acclimation responses to the drought and recovery cycles
were seen. The finding that gs, Amax and Γ reacted to drought
and rewatering in the first two cycles, but not in the third and
most severe drought cycle, indicates that these variables exhibited decreasing sensitivity to drought (Figures 1 and 2). It is
possible that stomatal sensitivity to drought decreased with
both seedling age and drought history. Although a reduction in
stomatal sensitivity with age has been reported for orange trees
(Syvertsen et al. 1981), stomatal sensitivity to drought usually
increases with age, especially in agricultural species (Field
1987).
A persistent difference between the control and recovering
seedlings 7 days after drought relief was observed for gs, Γ, Km
and Vrubisco (Figures 1 and 2, and Table 3). However, only gs
showed a consistent difference over the three cycles. The
responses of the other mesophyll photosynthesis variables
were either significant only in the second cycle (Km and
Vrubisco), or inconsistent in trend (Γ), possibly due to measurement error. In general, mesophyll photosynthetic function, as
reflected by mesophyll conductance, was largely independent
of water stress. In a broad range of species, stomatal closure
occurs before mesophyll photosynthetic function is greatly
affected by water stress (Gollan et al. 1985, Teskey et al. 1986,
Kirschbaum 1987, Grieu et al. 1988, Grossnickle and Russell
1991). In several studies, mesophyll photosynthetic capacity
under saturating CO2 showed little response to dehydration at
relative water contents as low as 50--70%, which is beyond the
usual turgor loss point (Kaiser 1987, Cornic et al. 1989, Epron
and Dreyer 1990).
It is possible that we observed only an apparent acclimation.
Drought stress treatments can suspend or slow down the normal process of photosynthetic development (Ludlow and Ng
1974). During the recovery periods between droughts, the
ontogenetic trends in gs, Γ, Amax and Vrubisco continued at
roughly the same rates in both control and drought-stressed
seedlings (cf. slopes in Figures 1 and 2). The apparent acclimation response could, therefore, be due to an offset in the
development curves of control and drought-stressed seedlings
caused by the slowing or cessation of development during the
drought periods.
62
STEWART, ZINE EL ABIDINE AND BERNIER
Photosynthetic limitation and drought resistance
The finding that stomatal limitation was less than mesophyll
limitation in black spruce has also been observed in other
conifers (Teskey et al. 1986, Grieu et al. 1988, Seiler and
Cazell 1990, Grossnickle and Russell 1991) and in C3 plants in
general (Bunce 1977, Briggs et al. 1986, Ni and Pallardy
1992). The relative stomatal limitation at the beginning of the
experiment was lower than most reported values; however,
stomatal limitation increased with seedling age and was similar to values found in other black spruce trees (calculated from
data in Macdonald and Lieffers 1990, Greenway et al. 1992).
The functional consequences of the drought-insensitivity of
mesophyll photosynthetic processes combined with the small
relative stomatal limitation of photosynthesis are illustrated by
plotting Anet against ci, as determined at the operating point (ca
= 350 µmol mol −1, Figure 3). For all seedlings pooled, there
was a poor correlation between Anet and ci (R2 = 0.019). However, when the data were separated into well-watered (including recovering seedlings) and drought-stressed subsets, the
regressions explained 45 and 31% of the variation, respectively. In drought-stressed seedlings, photosynthesis decreased
with decreasing ci as the stomata closed, whereas in well-watered seedlings the relationship was negative. The photosynthetic rate largely determined ci and stomatal conductance had
little influence. Thus, contrary to observations on several species by Wong et al. (1979), photosynthetic rate in our seedlings
was largely uncoupled from stomatal behavior, except during
periods of maximum drought when stomatal conductance was
very low.
In cases of heterogeneous stomatal closure, the calculated
Figure 3. The relationship between net photosynthetic rate (Anet) and
intercellular CO2 concentration (ci) in black spruce seedlings, calculated for an ambient CO2 concentration of 350 µmol mol −1. Circles are
control seedlings, triangles are seedlings under drought stress, and
squares are seedlings recovering from drought. Open squares and
triangles are seedlings in the middle of their respective recovery and
drought phases; filled symbols are seedlings at the ends of their
respective recovery and drought phases. Regression lines shown are
for control and recovering seedlings: Anet = 336.9 − 0.941ci (R2 =
0.311), and for drought-treated seedlings: Anet = −39.39 + 0.407ci (R2
= 0.447).
values of ci are higher than the real values, which can give the
appearance of mesophyll photosynthesis decreasing along
with stomatal conductance. The lack of such a coordinated
response to drought stress in our experiment supports our
assumption that heterogeneous stomatal closure did not occur.
Although there have been no reports of heterogeneous stomatal closure in conifers, we minimized the probability of
patchy stomatal closure by taking measurements soon after full
light exposure in high humidity on homobaric leaves that had
been exposed to a slowly increasing drought stress.
Gollan et al. (1985) postulated that the ability to maintain
constant mesophyll photosynthetic processes during water
stress, where photosynthesis is reduced only by a reduction in
stomatal conductance, is characteristic of a tolerance to soil
drought. In black spruce, mesophyll response was negligible
under drought conditions that produced soil water potentials of
−1.5 MPa. Limited mesophyll response to drought has been
noted in several species including Abies bornmuelleriana
Matt. (−1.0 MPa, Guehl et al. 1991), Eucalyptus pauciflora
Sieb. ex Spreng. (Kirschbaum 1987), Pseudotsuga macrocarpa (Torr.) Mayr (−1.5 MPa) and Pseudotsuga menziesii
(Mirb.) Franco (−1.9 MPa, Grieu et al. 1988). In Cedrus
atlantica Manetti, the entire drought response was due to
changes in mesophyll processes (Grieu et al. 1988, Guehl et al.
1991).
The drought resistance strategy of black spruce seedlings
appears to be one of maximizing, rather than optimizing,
photosynthetic rate (Tan et al. 1992). Under drought conditions, when stomatal closure limits CO2 uptake as well as water
loss, photosynthetic efficiency remains relatively unaffected.
Maximizing growth, particularly of the roots, is important in
establishment of young seedlings (van den Driessche 1987,
Burdett 1990). Although high mesophyll photosynthetic capacity may be of little value during severe droughts when
stomata close completely, during moderate droughts the maintenance of photosynthetic capability may improve the overall
net carbon gain and growth of black spruce seedlings.
We were unable to demonstrate unequivocally that the
drought treatments induced true active acclimation rather than
delaying the ontogenetic processes in black spruce seedlings.
However, our results show that photosynthetic capacity remained relatively unaffected by drought treatments causing
predawn water potentials of −1.5 MPa, although stomatal conductance was greatly reduced under such conditions. Stomatal
conductance accounted for less than 20% of photosynthetic
limitation under most conditions. Based on the variation in
photosynthetic capacity (Amax) and efficiency (Vrubisco) found
among black spruce seedlings, and the limited degree of control of photosynthetic rate by the stomata, we conclude that it
would be more effective to select for high photosynthetic
capacity than for reduced stomatal sensitivity when breeding
for increased drought resistance in black spruce (cf. Lauer and
Boyer 1992).
Acknowledgments
We thank Pierre Davignon for technical assistance, and Gilles Robitaille for the loan of equipment. Support for this research was provided
PHOTOSYNTHETIC LIMITATION BY DROUGHT
by Forestry Canada, Quebec Region, Research Project CFL-44. A
Natural Sciences and Engineering Research Council of Canada Forestry Postdoctoral Fellowship awarded to JDS is gratefully acknowledged. Dr. André Plamondon contributed greatly to the realization of
this postdoctoral position and the research carried out during its
tenure.
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© 1994 Heron Publishing----Victoria, Canada
Stomatal and mesophyll limitations of photosynthesis in black spruce
seedlings during multiple cycles of drought
J. D. STEWART,1,2 A. ZINE EL ABIDINE2 and P. Y. BERNIER1
1
Forestry Canada, Quebec Region, C.P. 3800, Sainte-Foy, Quebec G1V 4C7, Canada
2
Centre de Recherche en Biologie Forestière, Faculté de Foresterie et de Géomatique, Université Laval, Sainte-Foy, Québec G1K 7P4, Canada
Received June 7, 1993
Summary Container-grown black spruce (Picea mariana
(Mill.) B.S.P.) seedlings were planted in trays containing a sand
and peat mixture, and placed in a climate-controlled greenhouse. One group of seedlings was kept well-watered, and
another group was subjected to three cycles of drought. Gas
exchange analysis showed that mesophyll photosynthetic function was largely unimpaired by drought. In contrast, stomatal
conductance was sensitive to drought, although it became less
sensitive with each drought cycle. Both stomatal and mesophyll conductances increased with time in control and droughtstressed seedlings, but mesophyll conductance increased with
time more rapidly than did stomatal conductance. Limitation
of photosynthetic rate was dominated by the mesophyll. In
control seedlings, relative stomatal limitation increased from 6
to 16% by the end of the experiment. In drought-stressed
seedlings, relative stomatal limitation of photosynthesis
reached 40% during the first drought, but decreased to near
control values immediately after rewatering. Because the third,
most severe drought had only a minor effect on stomatal
conductance, relative stomatal limitation of photosynthesis
was similar to that in control seedlings by the end of the
experiment. Inhibition of ontogenetic change during drought
stress may be responsible for the apparent acclimation of
mesophyll photosynthetic processes. We conclude that it would
be more effective to select for high photosynthetic capacity
than for reduced stomatal sensitivity when breeding for increased drought resistance in black spruce seedlings.
Keywords: acclimation, container stock, drought resistance,
mesophyll conductance, Picea mariana, stomatal limitation,
stomatal sensitivity.
of conifer seedlings, any physiological or morphological adjustment that ameliorates the growth reduction due to water
stress will improve their rate of establishment.
The growth of a plant depends on a series of physical and
biochemical processes that transfer CO2 from the surrounding
air into organic molecules, and, for this reason, much work on
acclimation to water stress in trees has focused on the role of
stomata in limiting gas exchange in response to stress (e.g.,
Unterscheutz et al. 1974, Seiler 1985, van den Driessche
1991). Because mesophyll processes involved in the transfer of
CO2 into the chloroplasts and CO2 fixation may also be limiting during drought stress, there have been several studies of
photosynthetic as well as stomatal acclimation to water stress
in trees (e.g., Seiler and Johnson 1985, 1988, Walters and
Reich 1989). However, few studies have examined the relative
contributions of stomatal and nonstomatal acclimation to net
photosynthetic acclimation to drought in trees (Epron and
Dreyer 1990, Seiler and Cazell 1990) and nonwoody plants
(Hutmacher and Krieg 1983, Matthews and Boyer 1984, Cornic et al. 1987).
To identify the traits that contribute most to drought acclimation in black spruce seedlings, we subjected seedlings to
three cycles of drought and measured their photosynthetic
responses to CO2 during the drought and recovery periods. The
objectives of this study were (a) to test for acclimation to
drought stress in net photosynthesis of black spruce seedlings
over three drought cycles, (b) to determine the relative stomatal and nonstomatal contributions to the drought response,
and (c) to identify the mesophyll processes, if any, involved in
acclimation.
Materials and methods
Introduction
Young conifer seedlings are subject to a variety of environmental stresses when they are planted on clear-cut sites. Of
these stresses, drought is perhaps the most prevalent (Burdett
1990). Even in the boreal forest, a decrease in water content of
the surface horizons of the soil can subject shallow-rooted,
newly planted seedlings to severe water stress. Seedlings that
can acclimate to the first drought may be better able to survive
subsequent droughts. Because water stress inhibits the growth
Seedling growing conditions
Black spruce seeds from a lowland Quebec population were
planted in nursery containers (Rigipot, IPL, St-Damien, Quebec) with 50 ml cavities filled with a 3/1 (v/v) peat/vermiculite
medium. Seedlings were raised under standard conditions in a
commercial greenhouse for one growing season, hardened
outside and then placed in black plastic bags in a coldroom at
2 °C for 3 months.
58
STEWART, ZINE EL ABIDINE AND BERNIER
In January, the seedlings were moved to a greenhouse and
transplanted from the nursery containers to 15 × 60 cm trays,
each containing 10 seedlings. The trays were filled to a depth
of 10 cm with a 3/1 (v/v) fine sand/peat mixture. Trays were
watered daily by an automatic overhead watering boom
equipped with fine spray nozzles. Seedlings were fertilized
using the watering system twice per week, with each tray
receiving approximately 47 mg N,P,K (20-20-20) and 1.5 mg
MgSO4. Greenhouse day/night temperatures were 19--24/11-20 °C, and day/night relative humidity was 20--90/65--100%;
both varied with external weather conditions. The seedlings
were maintained in a 16-h photoperiod using high pressure
sodium vapor lamps (Lumalux GTE, Sylvania Canada Ltd.)
that provided 120 µmol m −2 s −1 PAR at seedling level. Tray
positions on the bench were randomized twice a week.
Bud burst occurred in early February, and the seedlings were
held under constant conditions until shoot elongation was
completed. At the beginning of the treatment period, seedlings
averaged 14.0 cm in height and 1.7 cm in diameter.
Cyclic drought and recovery treatment
Beginning March 11, the cyclic drought and recovery treatment was initiated by withholding water from the seedlings.
Control seedlings continued to be watered daily by hand.
Drought was allowed to progress until turgor loss (wilting)
became visible in the current shoots of some seedlings, whereupon the drought was terminated. Three such drought episodes
lasting 10, 10 and 8 days, respectively, were completed. Each
drought period was followed by a 7-day recovery period,
during which all seedlings were watered daily and fertilized as
described above. Each pair of drought and recovery periods
comprised a drought treatment cycle, and the three cycles
occupied 49 days. Soil water potential (Ψsoil) was measured
with calibrated gypsum blocks (Soilmoisture Equipment Corporation, Santa Barbara, CA), and predawn xylem water potential (Ψx) was measured with a pressure chamber (PMS
Instruments, Corvallis, OR) in neighboring nonexperimental
trays. The severity of the droughts as measured by soil and
predawn xylem water potentials is shown in Table 1 (see also
Zine El Abidine et al. 1995).
At intervals during the drought and recovery cycles, one
seedling from each of four trays was selected at random for gas
exchange measurements. Each seedling was excavated and
placed in a Ray Leach ‘‘Conetainer’’ (Stuewe and Sons, Corvallis, OR) in a controlled environment chamber. On each
measurement day, all four seedlings were either from the
control group or from the treatment group.
The response of net photosynthesis (Anet) to intercellular
CO2 concentration (ci), i.e., the A/ci relationship, was measured
by the nonsteady state method (Davis et al. 1987, McDermit et
al. 1989) in a closed-circuit gas analysis system (LI-6200,
Li-Cor Inc., Lincoln, NB). To standardize the measurement
process, the seedlings were placed in a controlled environment
chamber and kept in the dark in a loosely closed box until
needed. The concentration of CO2 in the chamber was elevated
as a result of respiration by the observer and was crudely
regulated to between 600 and 700 µmol mol −1. Water-cooled
high pressure sodium and mercury lamps were suspended
above the cuvette and provided 1000 µmol m 2 s −1 PAR at the
level of the cuvette. For additional heat dissipation, a transparent acrylic tray filled with water was placed between the lamp
and the cuvette. A fan circulated ambient air around the cuvette
to increase convective cooling. Each seedling was allowed to
adjust to the measurement light conditions for 30 min before
being placed in the 4-l cuvette. Once the seedling was sealed
in the gas exchange cuvette, the relative humidity was raised
to 85% by pumping ambient air through a gasing wash bottle,
filled with water at room temperature, into the gas exchange
system in open mode. The high humidity represented the upper
calibration limit of the humidity sensor (personal communication, J.M. Welles, Li-Cor Inc., Lincoln, NB) and was used to
avoid stomatal closure in response to high VPD in the waterstressed seedlings (Li-Cor 1991), which would have prevented
the gas exchange analysis and is known to provoke heterogeneous stomatal closure in some species. After closure, the
system was equilibrated under the measurement conditions for
20 to 30 min before measurements were taken.
Gas exchange measurements were taken continuously as the
seedling depleted the CO2 in the closed system. Observations
of CO2 and water fluxes were averaged for every 10 µmol
mol −1 change in CO2 concentration. Under severe water stress,
some seedlings had completely closed stomata, and the gas
exchange was too slow to be measured. These seedlings were
not included in the analysis.
Gas exchange parameters calculated were Anet, ci, CO2 compensation point (Γ), transpiration rate (E) and stomatal conductance to water vapor (gs). The program of the LI-6200 was
Table 1. Midday soil water potentials (Ψsoil) and predawn xylem water potentials (Ψx) measured at the end of each drought period before
rewatering. Means ± SE and number of observations are presented. All four soil water potentials measured at the end of the third drought were at
or beyond the range of the gypsum resistance blocks used.
Variable
Drought
Midday soil (MPa)
Treatment
Predawn
Control
Treatment
x (MPa)
1
2
3
−1.18 ± 0.520 (3)
−0.41 ± 0.139 (4)
> −2.00 * (4)
−0.40 ± 0.020 (4)
−1.17 ± 0.091 (15)
−0.75 ± 0.052 (16)
−0.98 ± 0.153 (16)
−0.59 ± 0.034 (16)
−2.62 ± 0.162 (16)
PHOTOSYNTHETIC LIMITATION BY DROUGHT
altered to account for the leak rate of the system as described
in Li-Cor (1991). When the CO2 depletion rate slowed in the
lower part of the curve, CO2 was removed by opening the CO2
scrubbing circuit of the LI-6200 for 45 s, followed by a 5-min
equilibration time before logging of observations resumed.
The average change in CO2 concentration per unit time during
the scrub cycle was similar to that during the continuous upper
portion of the curve. Ambient CO2 concentration used for leak
rate corrections was measured immediately after finishing a
curve. The needles were dried for 48 h in a forced air oven at
70 °C, and dry weights of new and old needles were determined on cooled samples.
Curve-fitting and parameter calculations
Various nonlinear functions were fitted to the CO2 response
(A/ci) data. Two three-parameter functions, the monomolecular
(or Mitscherlich) function and the rectangular hyperbolic function, did not produce satisfactory fits. The four-parameter
nonrectangular hyperbola suggested by Jones (1983) gave the
best fit to the data:
Anet =
Amax (ci − Γ − Anet Ri)
.
Km + ci − Γ − Anet Ri
(1)
The fitted parameters (Km, Amax, Γ and Ri) of the hyperbolic
function correspond to physiological properties of the photosynthetic system. The symbol Amax is the CO2 saturated rate of
net photosynthesis and represents the maximum rate of RuBP
regeneration by the Calvin cycle (Farquhar et al. 1980, von
Caemmerer and Farquhar 1981). The symbol Γ is the CO2
compensation point, which is primarily determined by photorespiration rates. The symbol Km is a constant for Rubisco
affinity for CO2, but it may also be influenced by the light
reactions (Jones 1983). The symbol Ri is a measure of the
resistance to liquid phase transport of CO2 within the mesophyll, but may also be determined by the activity of the carboxylase which may be acting well below its maximum rate in
the lower, near-linear portion of the response curve (Jones
1983). The carboxylation efficiency of Rubisco associated
with changes in concentration or activity (Vrubisco) was calculated from the first derivative of the function, evaluated at A =
0, and is closely related to 1/Ri.
Stomatal and mesophyll conductances to CO2 for an atmospheric concentration of 350 µmol mol −1 were obtained from
the response curve data following the procedure described by
Jones (1985). Stomatal conductance (gs) defined the slope of
the supply curve connecting ca with ci on the demand curve.
Mesophyll conductance (gm) was calculated as the slope of the
demand (A/ci) curve at its intersection with the supply curve.
Stomatal limitation of photosynthesis (Ls) was calculated as in
Jones (1985, Equation 17):
Ls =
1/gs
.
1/gs + 1/gm
(2)
59
Statistical analysis
For the control seedlings, the calculated variables were tested
for linear and quadratic responses over the four control measurement dates. Analyses of variance and a priori contrasts
were performed to assess (a) the differences in response among
the three drought cycles, and (b) the effect of the drought and
recovery treatment versus control conditions. Before the
analysis of variance, data variables were transformed as necessary to uphold the assumptions of normality and heteroscedasticity for the analysis of variance. No transformation was
required for Amax, Vrubisco and gs. All analyses were performed
using the General Linear Models procedure of SAS. Effects
were considered statistically significant when the probability
of a Type I error was 0.05 or less.
Results
Conductance to CO2 and photosynthetic limitation
Stomatal conductance of control seedlings increased with
seedling age, though only during Cycle 3 (Figure 1). Seedlings
recovering from drought showed a similar trend, but stomatal
conductance was generally lower in recovering seedlings than
in control seedlings, especially in Cycles 1 and 3 (Cycle 2
Figure 1. Panel (a) shows the responses of stomatal conductance
(circles) and mesophyll conductance (triangles) of black spruce seedlings to three cycles of drought and recovery. Panel (b) shows the
relative stomatal limitation of photosynthesis (Ls). Open symbols
represent control seedlings and closed symbols represent treatment
seedlings. Each point and bar represent the mean and SE of four
seedlings, except for the point marked by an asterisk, which represents
only one seedling. The timing of the droughts is indicated by the solid
horizontal bars at the bottom of the figure.
60
STEWART, ZINE EL ABIDINE AND BERNIER
produced only a mild stress; Table 1). Drought caused a large
decrease in stomatal conductance, but recovery following rewatering was rapid. The drought-induced reduction in stomatal
conductance was least in Cycle 3, even though this was the
most severe drought.
Mesophyll conductance increased linearly with age in control (Table 2) and drought-stressed seedlings (Figure 1), and
there was no significant difference between control and
drought-stressed seedlings (Table 3).
In control seedlings, the relative stomatal limitation of photosynthesis increased linearly with age (Table 2), rising from
6% at the beginning of the experiment to about 16% at the end
(Figure 1). Stomatal limitation increased to about 40% during
the period of maximum drought stress in Cycle 1, but the effect
was much less during the drought periods in Cycles 2 and 3
(Figure 1). Relative stomatal limitation quickly declined to
near control values on rewatering, and there were no significant differences between recovering and control seedlings in
Cycles 2 and 3 (Table 3). Overall, the rate of photosynthesis
was less affected by stomatal conductance than by mesophyll
conductance.
Gas exchange parameters
Most of the gas exchange parameters measured in control
seedlings increased with time (Table 2 and Figure 2), whereas
the effect of drought varied among the gas exchange parameters and drought cycles.
Gas exchange parameters of both control and recovering
seedlings showed increases mostly in Cycle 3 (Table 3); however, Γ also increased from Cycle 1 to Cycle 2. There was a
significant difference in Γ between control and recovering
seedlings late in Cycles 1 and 2 (Table 2 and Figure 2). By
Cycle 3, Γ was lower in recovering seedlings than in control
seedlings. Both control and recovering seedlings exhibited
similar Amax. Drought only affected Amax during Cycles 1 and 2
(Figure 2 and Table 3). The value of Km decreased with
drought, but tended to recover to values greater than those of
control seedlings on rewatering, but this effect was only statistically significant in Cycle 2 (Table 2 and Figure 2). There was
a significant difference in Vrubisco between control and recovering seedlings late in Cycle 2 (Table 2 and Figure 2). Drought
only affected Vrubisco during Cycle 2.
Discussion
Table 2. Regression analysis of the linear response of the measured
variables in control seedlings with respect to time. The parameter
estimates for intercept and slope, and the t-test probability that slope
is not equal to zero are presented.
Variable
Intercept
Slope
Probability
gs
gm
Ls
Amax
Γ
Km
Vrubisco
1.64
0.035
0.048
59.5
−0.089
4.45
0.261
0.0114
0.0049
0.0025
1.47
0.934
0.248
0.0046
0.070
0.001
0.002
0.002
0.001
0.002
0.001
Ontogenetic changes
As seedlings go through a series of drought cycles, they respond not only to water stress, but also to increasing age and
ontogenetic state. The A/ci curves of control seedlings showed
changes in photosynthetic properties over time. The values of
Amax, Vrubisco and Km increased with age (Figure 2), resulting in
an increase in mesophyll conductance with age (Figure 1). All
of these processes changed in concert, suggesting that this
ontogenetic response is a reflection of the increasing photosynthetic competence of the needles as they mature (Ludlow and
Wilson 1971, Field 1987).
The apparent trend in Γ may not be real, because values
Table 3. Statistical summary for gas exchange and photosynthetic parameters for control seedlings and treatment seedlings at the end of the
recovery periods following each drought. Probability values from a two-way ANOVA with contrasts are presented.
Effect
df
gs
gm
Cycle
Treatment
Cycle × treatment
2
1
2
0.002
0.004
0.441
0.001
0.498
0.673
Contrasts----Controls
Cycle 3 versus 1 + 2
Cycle 1 versus 2
2
1
0.012
0.423
Contrasts----Recovering
Cycle 3 versus 1 + 2
Cycle 1 versus 2
2
1
Amax
G
Km
Vrubisco
0.018
0.080
0.383
0.000
0.690
0.934
0.000
0.015
0.003
0.001
0.010
0.294
0.000
0.042
0.508
0.006
0.122
0.027
0.066
0.006
0.428
0.000
0.000
0.029
0.499
0.001
0.072
0.024
0.336
0.033
0.647
0.222
0.995
0.003
0.680
0.000
0.000
0.003
0.008
0.004
0.818
Contrasts----Controls versus recovering
Cycle 1
1
0.015
Cycle 2
1
0.382
Cycle 3
1
0.046
0.273
0.979
0.926
0.043
0.829
0.428
0.891
0.602
0.974
0.495
0.597
0.000
0.595
0.011
0.129
0.674
0.050
0.220
Error mean square
0.0268
0.304
556
44964
0.0221
0.00419
18
0.0720
Ls
PHOTOSYNTHETIC LIMITATION BY DROUGHT
61
Figure 2. The response of photosynthetic parameters of black
spruce seedlings to three cycles of
drought and recovery. Panels: (a)
CO2 compensation point, (b) affinity constant for carbon fixation,
(c) CO2 saturated maximum net
photosynthetic rate, and (d) maximum rate of carboxylation of
RuBP. The units for Vrubisco are
nmol CO2 g − 1 s − 1 per µmol
mol − 1 CO2. Open symbols represent control seedlings and closed
symbols represent treatment seedlings.
below 10 µmol mol −1 are not likely to occur in a C3 plant
(normal range is 30 to 70 µmol mol −1). The data points at the
bottom of the curve, which have the greatest influence on the
value of Γ, have a lower precision than those at the top of the
curve, due to the very low rates of gas exchange at low ca.
Unlike the other gas exchange parameters, the calculated values of Γ were sensitive to the form of equation used to fit the
data. The monomolecular function gave more realistic and
consistent values of Γ than the nonrectangular hyberbolic
function, but the overall fit was not as good, especially for the
upper portion of the curve.
Stomatal conductance increased with age. Relative stomatal
limitation of photosynthesis also increased with age, because
mesophyll conductance increased faster than stomatal conductance (Table 2 and Figure 2). Although both gs and gm increase
with age in most plants, the relative rates of increase vary
among species (e.g., Ludlow and Wilson 1971, Field 1987,
Mebrahtu and Hanover 1991). Consequently, the change in
relative stomatal limitation of photosynthesis can be positive,
negative or constant, depending on the species.
Acclimation
Acclimation in drought-stressed seedlings is manifested as
either a change in the degree of response to drought, or a
persistent difference between recovering seedlings and the
controls. Although the values of photosynthetic variables in
treated and control seedlings followed similar trends with
time, acclimation responses to the drought and recovery cycles
were seen. The finding that gs, Amax and Γ reacted to drought
and rewatering in the first two cycles, but not in the third and
most severe drought cycle, indicates that these variables exhibited decreasing sensitivity to drought (Figures 1 and 2). It is
possible that stomatal sensitivity to drought decreased with
both seedling age and drought history. Although a reduction in
stomatal sensitivity with age has been reported for orange trees
(Syvertsen et al. 1981), stomatal sensitivity to drought usually
increases with age, especially in agricultural species (Field
1987).
A persistent difference between the control and recovering
seedlings 7 days after drought relief was observed for gs, Γ, Km
and Vrubisco (Figures 1 and 2, and Table 3). However, only gs
showed a consistent difference over the three cycles. The
responses of the other mesophyll photosynthesis variables
were either significant only in the second cycle (Km and
Vrubisco), or inconsistent in trend (Γ), possibly due to measurement error. In general, mesophyll photosynthetic function, as
reflected by mesophyll conductance, was largely independent
of water stress. In a broad range of species, stomatal closure
occurs before mesophyll photosynthetic function is greatly
affected by water stress (Gollan et al. 1985, Teskey et al. 1986,
Kirschbaum 1987, Grieu et al. 1988, Grossnickle and Russell
1991). In several studies, mesophyll photosynthetic capacity
under saturating CO2 showed little response to dehydration at
relative water contents as low as 50--70%, which is beyond the
usual turgor loss point (Kaiser 1987, Cornic et al. 1989, Epron
and Dreyer 1990).
It is possible that we observed only an apparent acclimation.
Drought stress treatments can suspend or slow down the normal process of photosynthetic development (Ludlow and Ng
1974). During the recovery periods between droughts, the
ontogenetic trends in gs, Γ, Amax and Vrubisco continued at
roughly the same rates in both control and drought-stressed
seedlings (cf. slopes in Figures 1 and 2). The apparent acclimation response could, therefore, be due to an offset in the
development curves of control and drought-stressed seedlings
caused by the slowing or cessation of development during the
drought periods.
62
STEWART, ZINE EL ABIDINE AND BERNIER
Photosynthetic limitation and drought resistance
The finding that stomatal limitation was less than mesophyll
limitation in black spruce has also been observed in other
conifers (Teskey et al. 1986, Grieu et al. 1988, Seiler and
Cazell 1990, Grossnickle and Russell 1991) and in C3 plants in
general (Bunce 1977, Briggs et al. 1986, Ni and Pallardy
1992). The relative stomatal limitation at the beginning of the
experiment was lower than most reported values; however,
stomatal limitation increased with seedling age and was similar to values found in other black spruce trees (calculated from
data in Macdonald and Lieffers 1990, Greenway et al. 1992).
The functional consequences of the drought-insensitivity of
mesophyll photosynthetic processes combined with the small
relative stomatal limitation of photosynthesis are illustrated by
plotting Anet against ci, as determined at the operating point (ca
= 350 µmol mol −1, Figure 3). For all seedlings pooled, there
was a poor correlation between Anet and ci (R2 = 0.019). However, when the data were separated into well-watered (including recovering seedlings) and drought-stressed subsets, the
regressions explained 45 and 31% of the variation, respectively. In drought-stressed seedlings, photosynthesis decreased
with decreasing ci as the stomata closed, whereas in well-watered seedlings the relationship was negative. The photosynthetic rate largely determined ci and stomatal conductance had
little influence. Thus, contrary to observations on several species by Wong et al. (1979), photosynthetic rate in our seedlings
was largely uncoupled from stomatal behavior, except during
periods of maximum drought when stomatal conductance was
very low.
In cases of heterogeneous stomatal closure, the calculated
Figure 3. The relationship between net photosynthetic rate (Anet) and
intercellular CO2 concentration (ci) in black spruce seedlings, calculated for an ambient CO2 concentration of 350 µmol mol −1. Circles are
control seedlings, triangles are seedlings under drought stress, and
squares are seedlings recovering from drought. Open squares and
triangles are seedlings in the middle of their respective recovery and
drought phases; filled symbols are seedlings at the ends of their
respective recovery and drought phases. Regression lines shown are
for control and recovering seedlings: Anet = 336.9 − 0.941ci (R2 =
0.311), and for drought-treated seedlings: Anet = −39.39 + 0.407ci (R2
= 0.447).
values of ci are higher than the real values, which can give the
appearance of mesophyll photosynthesis decreasing along
with stomatal conductance. The lack of such a coordinated
response to drought stress in our experiment supports our
assumption that heterogeneous stomatal closure did not occur.
Although there have been no reports of heterogeneous stomatal closure in conifers, we minimized the probability of
patchy stomatal closure by taking measurements soon after full
light exposure in high humidity on homobaric leaves that had
been exposed to a slowly increasing drought stress.
Gollan et al. (1985) postulated that the ability to maintain
constant mesophyll photosynthetic processes during water
stress, where photosynthesis is reduced only by a reduction in
stomatal conductance, is characteristic of a tolerance to soil
drought. In black spruce, mesophyll response was negligible
under drought conditions that produced soil water potentials of
−1.5 MPa. Limited mesophyll response to drought has been
noted in several species including Abies bornmuelleriana
Matt. (−1.0 MPa, Guehl et al. 1991), Eucalyptus pauciflora
Sieb. ex Spreng. (Kirschbaum 1987), Pseudotsuga macrocarpa (Torr.) Mayr (−1.5 MPa) and Pseudotsuga menziesii
(Mirb.) Franco (−1.9 MPa, Grieu et al. 1988). In Cedrus
atlantica Manetti, the entire drought response was due to
changes in mesophyll processes (Grieu et al. 1988, Guehl et al.
1991).
The drought resistance strategy of black spruce seedlings
appears to be one of maximizing, rather than optimizing,
photosynthetic rate (Tan et al. 1992). Under drought conditions, when stomatal closure limits CO2 uptake as well as water
loss, photosynthetic efficiency remains relatively unaffected.
Maximizing growth, particularly of the roots, is important in
establishment of young seedlings (van den Driessche 1987,
Burdett 1990). Although high mesophyll photosynthetic capacity may be of little value during severe droughts when
stomata close completely, during moderate droughts the maintenance of photosynthetic capability may improve the overall
net carbon gain and growth of black spruce seedlings.
We were unable to demonstrate unequivocally that the
drought treatments induced true active acclimation rather than
delaying the ontogenetic processes in black spruce seedlings.
However, our results show that photosynthetic capacity remained relatively unaffected by drought treatments causing
predawn water potentials of −1.5 MPa, although stomatal conductance was greatly reduced under such conditions. Stomatal
conductance accounted for less than 20% of photosynthetic
limitation under most conditions. Based on the variation in
photosynthetic capacity (Amax) and efficiency (Vrubisco) found
among black spruce seedlings, and the limited degree of control of photosynthetic rate by the stomata, we conclude that it
would be more effective to select for high photosynthetic
capacity than for reduced stomatal sensitivity when breeding
for increased drought resistance in black spruce (cf. Lauer and
Boyer 1992).
Acknowledgments
We thank Pierre Davignon for technical assistance, and Gilles Robitaille for the loan of equipment. Support for this research was provided
PHOTOSYNTHETIC LIMITATION BY DROUGHT
by Forestry Canada, Quebec Region, Research Project CFL-44. A
Natural Sciences and Engineering Research Council of Canada Forestry Postdoctoral Fellowship awarded to JDS is gratefully acknowledged. Dr. André Plamondon contributed greatly to the realization of
this postdoctoral position and the research carried out during its
tenure.
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