Copyright ª Physiologia Plantarum 2007, ISSN 0031-9317
Physiologia Plantarum 130: 207–217. 2007
Combined drought and episodic freezing effects on
seedlings of low- and high-elevation subspecies of
sagebrush (Artemisia tridentata)
Susan C. Lambrechta,*, Anne K. Shattuckb and Michael E. Loikb
a
Department of Biological Sciences and the Center for Biodiversity, San José State University, San José, CA 95192, USA
Department of Environmental Studies, University of California, Santa Cruz, Santa Cruz, CA 95064, USA
b
Correspondence
*Corresponding author,
e-mail: [email protected]
Received 13 December 2006; revised 25
January 2007
doi: 10.1111/j.1399-3054.2007.00904.x
Big sagebrush (Artemisia tridentata) is a dominant shrub throughout much of
the arid western United States. Several recognized subspecies differ in
physiology, morphology and in their distribution in relation to soil water
availability. While several studies have compared mature individuals of these
subspecies, there is little information on seedling physiological tolerance to
physical stresses. Understanding seedling physiology is essential for predicting
how species may respond to changes in temperature and precipitation regimes.
Our objective was to examine the drought and freezing tolerance of seedlings
of two A. tridentata subspecies: ssp. tridentata, which is found in low-elevation
dry sites, and ssp. vaseyana, found in higher, moister sites. We examined
growth, gas exchange and quantum yield of chlorophyll a fluorescence from
photosystem II (PSII) for seedlings grown in a greenhouse and exposed to two
different levels of moisture availability in combination with a simulated
growing season freezing event. We found that ssp. tridentata possessed several
drought-tolerant characteristics, such as rapid growth rates, thick leaves and
low stomatal conductance. Both subspecies shared similar physiological
tolerance to the low-moisture treatment but experienced relatively more stress
under the freezing treatment. Subspecies vaseyana was more sensitive to
freezing when grown with low soil moisture, resulting in reduced stomatal
conductance and PSII quantum yield. In contrast, the low-soil moisture
treatment did not increase the susceptibility of ssp. tridentata to freezing. These
results demonstrate that drought tolerance may be an important trait for
seedlings of A. tridentata, but stress because of freezing damage of seedlings
may limit the distribution of the species.
Introduction
Big sagebrush (Artemisia tridentata Nutt., Asteraceae) is
the most common and widespread shrub species of the
western United States, dominating 36.5 million ha,
primarily in the Great Basin Desert (Miller et al. 1986).
Across its range, it can be found in a variety of habitats,
from valleys to mountain slopes and alpine benches
(Shultz 1986b). There are at least four recognized subspecies of A. tridentata that partition the range of the
species based on elevation and soil moisture availability
(Miller et al. 1986, West 1988). Of the two most common
subspecies, ssp. vaseyana (mountain big sagebrush) is
found on gravelly soils of mountain slopes, from approximately 1100–2700 masl (Shultz 1986b). In contrast,
Abbreviations – HID, high-intensity discharge; PAR, photosynthetically active radiation; PSII, photosystem II; RGR, relative growth
rate; SLA, specific leaf area; WUE, water-use efficiency.
Physiol. Plant. 130, 2007
207
subspecies tridentata (Basin big sagebrush) is found on
well-drained, xeric soils of lower elevation valleys and
foothills up to 2000 masl (Shultz 1986b, West 1988). In
the habitat of ssp. vaseyana, annual precipitation can be
two to three times greater than that found at the lower
elevation habitats of ssp. tridentata (Miller et al. 1986,
West 1988). Furthermore, soil moisture availability is
greater and soil temperatures are lower in the range of ssp.
vaseyana than that of ssp. tridentata (Miller et al. 1986,
West 1988). The subspecies can hybridize at intermediate
elevations (McArthur et al. 1998).
Whereas all A. tridentata subspecies appear to be well
adapted to arid environments, some have morphological
and physiological characteristics that enable them to
succeed in more xeric environments than others. In particular, ssp. tridentata has a suite of characters that promote greater water conservation as compared with ssp.
vaseyana, such as thicker leaves, higher water-use efficiency (WUE) and greater stomatal resistance to water
vapor loss (Frank et al. 1986, Kolb and Sperry 1999a,
Shultz 1986a). Subspecies tridentata also exhibits a suite
of traits that enable it to function at lower soil moisture
availabilities than ssp. vaseyana, such as narrower stem
and leaf vessel diameter, which confers greater resistance
to cavitation, and greater growth rates, which presumably
enables ssp. tridentata to grow rapidly before soil moisture becomes too limiting during summer drought (Frank
et al. 1986, Shultz 1986a, Welch and McArthur 1986).
Many of these differences have been observed in commongarden studies, suggesting that they are heritable and
under selection (Barker and McKell 1986, Booth et al.
1990, McArthur and Welch 1982, McArthur et al. 1998).
Therefore, soil moisture availability is an important environmental factor contributing to the geographic and taxonomic segregation of the subspecies of A. tridentata.
Given the different elevational distributions of each
A. tridentata subspecies, low temperatures may be an additional environmental factor determining their ranges. In
particular, tolerance of seedlings to episodic low air temperature events may be an important factor in determining
subspecies distributions (Loik and Redar 2003). Diurnal
and seasonal temperature fluctuations in the Great Basin
Desert can be extreme, with the potential for subzero air
temperatures occurring on nearly any day of the year
(Beatley 1975, Hidy and Klieforth 1990, Smith and Nowak
1990). Furthermore, in cold desert environments, air temperatures near the soil surface are typically lower than and
experience greater diurnal fluctuations than the air 1 m
above (Nobel 1997). Therefore, the microenvironment in
which seedlings grow is often colder than that of nearby
mature plants. Moreover, seedlings are typically less tolerant of environmental extremes than are mature plants
(Boorse et al. 1998, Bruelheide and Heinemeyer 2002,
208
Hamann 2001, Pratt et al. 2005, Sakai and Larcher 1987).
Thinner tissues of young plants coupled with a higher
sensitivity to freezing can result in damaged membranes
and tissues, reduced physiological function and even death
(Sakai and Larcher 1987). As a result, the distribution of
several arid land plants appears limited by the survival of
seedlings in relation to freezing temperatures (Boorse et al.
1998, Franco and Nobel 1989, Jordan and Nobel 1979,
Loik and Redar 2003, Nardini et al. 1998). A recent study of
freezing tolerance of A. tridentata ssp. tridentata seedlings
revealed that the ability to acclimate to freezing temperatures increased with elevation of the source population
(Loik and Redar 2003). Given the differences in elevation
and temperature among A. tridentata subspecies ranges,
tolerance of freezing may also be an important factor
determining subspecies distributions.
The objective of this study was to compare the effects of
soil moisture availability in combination with a single
freezing event on growth and physiological performance
for seedlings of two A. tridentata subspecies: ssp.
tridentata and ssp. vaseyana. Prior studies that compared
physiological differences among A. tridentata subspecies,
such as resistance to cavitation, WUE and stomatal
resistance to water loss (e.g. Frank et al. 1986, Kolb and
Sperry 1999a, Shultz 1986a, Welch and McArthur 1986),
have compared mature plants. There is little known about
the physiological differences of seedlings. We used a soil
moisture control method to impose two different soil
water potential regimes on seedlings grown from seed in
a common-garden experiment in a greenhouse. Half of
these seedlings were then exposed to a single freezing
event, similar to that experienced during an episodic cold
snap late in the growing season. We assessed seedling
responses through measurements of growth, foliar gas
exchange and quantum yield of chlorophyll a fluorescence from photosystem II (PSII), as indicators of stress
tolerance. We hypothesized, given the distributional
limits of these subspecies, that ssp. tridentata would be
more tolerant of low soil moisture than ssp. vaseyana,
while ssp. vaseyana would be more tolerant of freezing.
We also hypothesized that plants grown under the
drought treatment would be more tolerant of freezing
than those grown under the high moisture treatment. That
is, we expected that plants exposed to the combination of
drought and freezing treatments would respond differently than they had to either of the treatments alone.
Materials and methods
Plant material
The two subspecies of A. tridentata used in this study
can be distinguished by differences in distribution,
Physiol. Plant. 130, 2007
morphological and physiological characters and ploidy
levels. While both subspecies can be either diploid or
tetraploid, ssp. vaseyana is more typically diploid while
ssp. tridentata is more commonly tetraploid (McArthur
et al. 1981). Seeds for ssp. vaseyana were obtained near
Poncha Pass, Saguache County, CO (approximately
2700 masl), while seeds for ssp. tridentata were obtained
from Uintah County, UT (approximately 1800 masl); each
of these locations included the single subspecies. Seeds
were kept in a cool (approximately 18C), dark storage
container for approximately 3 months prior to being
used in the experiment. Growth conditions are described
below.
Greenhouse experiment
The experiment was conducted in a rooftop greenhouse
on the campus of the University of California, Santa Cruz.
For the duration of the experiment, photoperiod was set to
14 h, using high-intensity discharge (HID) lamps with
metal halide bulbs. Average instantaneous daytime photosynthetically active radiation (PAR; 400–700 nm) was
approximately 1000 mmol m22 s21 in the horizontal
plane: PAR was maintained at this level by the HID lamps
on cloudy days. Day/night air temperatures were maintained at approximately 27/18C. Although these conditions do not exactly duplicate field conditions, they
approximate average, optimal growing season conditions
near the soil surface over the range of A. tridentata (Hidy
and Klieforth 1990). In a previous common-garden study
of seedlings of this species, we used a similar temperature
regimen (Loik and Redar 2003). When we shifted the
seedlings to cooler day/night air temperatures, we found
limited capacity for cold acclimation. Furthermore,
because freezing temperatures can occur on any night
of the year over the range of this species, seedlings may
not experience periods of appreciable low-temperature
acclimation prior to freezing events.
Drought was imposed by modifying a method described by Snow and Tingey (1985) and Fernández and
Reynolds (2000). This method enabled us to overcome
some common constraints of studying plant–soil water
interactions, including (1) distinguishing between plant
effects on – and responses to – soil water availability, (2)
distinguishing between drought-induced responses and
size-dependent plant responses to decreased soil water
availability (Fernández and Reynolds 2000) and (3)
technical difficulties of maintaining constant soil water
potential values from day-to-night and from day-to-day. In
this experiment, we placed open-bottom pots over
a porous medium that was placed in a reservoir of water
of a controlled depth (Fig. 1). With this procedure, soil
water potential within the pots is controlled at a uniform
Physiol. Plant. 130, 2007
P
B
F
W
Fig. 1. Schematic of the soil moisture controlling method modified from
Snow and Tingey (1985) and Fernández and Reynolds (2000). B, 90-l bins;
W, water depth; F, porous foam; P, open-bottom pots filled with sand:soil
mixture.
and repeatable level that is independent of plant size
(Fernández and Reynolds 2000, Saulescu et al. 1995,
Wookey et al. 1991).
Ten 90-l bins were made using 35-cm tall aluminum
flashing lined with 1-mm thick flexible plastic (B in
Fig. 1). The porous medium for conducting water was
a block of 25-cm tall florist’s foam (F in Fig. 1; SmithersOasis, Kent, OH). Within each of the 10 bins, six 15-cm
tall, 11-cm diameter pots (P in Fig. 1) were placed on the
foam. Each subspecies was planted in three pots in each
bin, for a total of six pots per bin. One pot per subspecies
was dedicated to the destructive sampling needed for
measurements of predawn water potential (see below).
The four remaining pots were used for all other measurements. Each pot was filled with a 4:1 mixture of coarse
sand and Pro-mix HP potting soil (Premier Horticulture,
Duval, QB, Canada). Seeds were sown directly onto
the soil surface in each pot and lightly covered with the
Pro-mix. Seedlings were thinned to three seedlings per
pot approximately 3 weeks after planting. The bottom of
each pot was covered with 0.04-mm nylon mesh to permit flow of water and air, while inhibiting root passage.
The 10 bins were assigned to five blocks, with one bin
in each block randomly assigned to one of two soil
moisture levels. Based on a pilot experiment, soil water
levels in the containers were maintained at a specific
hydraulic head (W in Fig. 1) in order to impose one of two
soil moisture levels (control and low moisture; Table 1).
These levels reflect average high- and low-soil moisture
levels typical for June and July at the sites of seed
collection for these two subspecies of A. tridentata (Black
and Mack 1986, Campbell and Harris 1977, Evans et al.
1990, Kolb and Sperry 1999a, 1999b, McArthur et al.
1998). The treatments were not imposed until 4 weeks
after seeds were planted to ensure seed germination and
root development. This delay also approximated field
conditions, in which the onset of seed germination and
early seedling growth occurs in spring when soil moisture
209
Table 1. Average predawn leaf water potential values (1 SE) in each of
the treatments. Values shown are the average of measurements of both
subspecies in each of the blocks (n ¼ 10 for each moisture level).
Predawn CL (MPa)
Predawn CL (MPa)
Treatment
Water
depth (cm)
Week 6
Week 12
Control
Low moisture
10
2.5
20.50 (0.01)
22.65 (0.04)
20.53 (0.02)
22.70 (0.02)
is most available (Smith and Nowak 1990, Young et al.
1988). Average leaf area per plant was not significantly
different at the end of this pretreatment period (t ¼ 0.26,
P ¼ 0.79, n ¼ 40).
Twelve weeks into the experiment (8 weeks after
drought imposition), an episodic freezing treatment was
imposed on one randomly selected pot per subspecies
from each bin. This imposed freezing event occurred at
the average end of the freeze-free period at the seed
source locations (Hidy and Klieforth 1990, Smith and
Nowak 1990). Each pot was removed and first placed in
a dark cooler (approximately 10C) for 60 min to be
exposed to gradually declining temperatures. Then, pots
were placed in a dark freezer in which temperatures were
gradually reduced over an hour until reaching a low of
25C, at which plants were held for an hour. Following
the freezing treatment, plants warmed in the greenhouse
at a rate of approximately 3C per hour until reaching
20C before measurements were made (see below). In
a field setting, temperatures would decline slowly over
a greater length of time than that which we simulated in
this study, possibly enabling some acclimation to
temperature change in the plants; however, most
greenhouse-based studies show a halftime for acclimation of 3 days (Larcher 2001). We were unable to draw
down temperatures over a longer period of time because
of concern of significantly reducing soil moisture levels in
the pots while removed from the greenhouse. Soil
moisture did not change significantly during the duration
of the freezing treatment (t ¼ 0.66, P ¼ 0.29, n ¼ 20), as
measured with a WP4 soil moisture meter (Decagon
Devices, Pullman, WA). Following the imposition of the
freezing treatment, plants were returned to the greenhouse conditions described above and allowed to grow
for approximately 3 weeks before final harvesting and
measurements were made (see below).
Measurements and analyses
Beginning with the drought imposition and continuing
approximately every 2 weeks thereafter, whole-plant leaf
area was estimated for two plants of each subspecies
210
within each of the bins. All leaves on each of the plants
were traced onto a piece of paper. Leaf traces were then
blackened (to enhance contrast), and leaf area was
measured with an area meter (CI-202, CID, Inc., Camas,
WA). Relative growth rate (RGR) was calculated from
these measurements of leaf area. Because RGRs can be
expected to change over time (Hunt 1982), two sets of
growth comparisons were made over two different time
scales. RGRs measured between weeks 4 and 12 (before
the imposition of the freezing treatment) were compared
between plants grown under both moisture treatments,
but without freezing. RGRs were then compared among
all treatments for the period of time after the freezing
treatment (i.e. between weeks 13 and 16). At the conclusion of the experiment, two mid-stem leaves were
harvested from each of the plants. The leaves were dried
in an oven at 70C for 24 h, then weighed and analyzed
with the leaf area meter. Specific leaf area (SLA) was
calculated as the average ratio of leaf area to leaf mass for
each plant. Values of SLA were not compared between
temperature treatments because growth substantially
slowed or stopped altogether for plants exposed to the
episodic freezing treatment and, therefore, it was not
possible to measure temperature-induced differences in
SLA. The entire shoot and root system of each of the plants
were also harvested, dried and weighed to obtain final
plant biomass measurements.
Following the imposition of the freezing treatment,
chlorophyll a fluorescence from PSII and photosynthetic
gas exchange measurements were made on fully elongated leaves of plants in each of the treatments (i.e. five
plants per each of the four moisture/freezing combination
treatments). Chlorophyll fluorescence was measured as
maximum quantum yield of PSII (Fv/Fm) for dark-adapted
plants, where Fv/Fm is a measure of the fraction of
absorbed photons used for photosynthesis in a darkadapted leaf and is a sensitive indicator of temperatureinduced stress for A. tridentata (Baker et al. 1988, Krause
et al. 1988, Loik and Harte 1996, Loik and Redar 2003,
Loik et al. 2004b). After the freezing treatment was
completed and following dark adaptation (20 min),
which occurred after greenhouse lights were turned off,
Fv/Fm was measured using a pulse amplitude-modulated
leaf chamber fluorometer (Model 6400-40, Li-Cor, Inc.,
Lincoln, NE) integrated with a portable, open-mode
photosynthesis system (Li-Cor Model 6400). All fluorescence and gas exchange measurements were made on the
first full-sized leaf below the apical meristem. To generate
Fm, the excitation flash duration was 0.8 s, with an
intensity setting of 8; the measurement modulation was
20 kHz, and the fluorescence signal was filtered at 50 Hz.
Sensitivity of PSII was also assessed based on the lightadapted maximum quantum yield (FPSII), where FPSII is
Physiol. Plant. 130, 2007
a measure of the fraction of absorbed photons that are
used for photosynthesis in light-adapted leaves (Baker
et al. 1988, Krause et al. 1988). Measurements of FPSII
were made using the same excitation and measurement
settings as above and were conducted at the same time as
the gas exchange measurements for each treatment.
Photosynthesis (A), transpiration (E) and stomatal conductance to water vapor (gs) were measured on one fully
elongated leaf of all plants between 07:00 and 11:30 h
local time using the LI-6400. These measurements were
completed in the 3 days following the freezing treatment.
Cuvette PAR, leaf-to-air vapor pressure deficit and pCO2
were maintained at ambient (greenhouse) levels during
measurements. After leaves were placed in the cuvette,
dark-adapted and all other values had stabilized; three
measurements were logged at 10-s intervals. An average of
these measurements was used for analyses. These measurements were area corrected, using leaf area measured on the
trace of the leaves placed in the cuvette, as described above.
Instantaneous WUE was calculated as the ratio of A to E.
Using the pots reserved for destructive water potential
measurements (one pot per subspecies in each bin),
predawn (04:00 h) leaf water potential (Cleaf) was measured twice during the experiment for both subspecies
with a Scholander-type pressure chamber (PMS Instruments, Corvallis, OR).
Treatment effects were tested using blocked ANOVA with
a generalized linear model in SYSTAT (SPSS, Inc., San Jose,
CA) using the paired bins as the blocks, as previously
described. The treatment effects tested in the model were
those of subspecies, moisture and freezing treatments as
well as all interactions. Post hoc tests were analyzed with
Tukey pairwise comparisons. Moisture treatment and
subspecies effects on Cleaf values were tested with twoway ANOVA. Assumptions of normality and homogeneity of
variance were examined with visual plots of the data and
residuals. For all analyses, we used an a ¼ 0.05 level of
significance.
Results
Predawn Cleaf values were consistently different between
the moisture treatments (Table 1; two-way ANOVA F ¼
188.83; P < 0.001). There were no differences, however,
in Cleaf values measured on the different subspecies
within each of the treatments (two-way ANOVA F ¼ 1.24;
P ¼ 0.29).
Parameters of plant size and foliar growth varied among
the treatments and subspecies (Tables 2 and 3). Final leaf
area was approximately 40% greater for ssp. tridentata
than ssp. vaseyana and was lower for both subspecies
under both the low moisture (approximately 70%) and
following the freezing (approximately 70%) treatment
Physiol. Plant. 130, 2007
(Fig. 2, Table 3). For both subspecies under high moisture,
plants exposed to the freezing treatment did not grow
further and accumulate as much leaf area as the other
plants, but only significantly so for ssp. tridentata (Tukey
P ¼ 0.001 for ssp. tridentata and P ¼ 0.08 for ssp.
vaseyana). These differences in leaf area between the
subspecies were because of the 60% higher leaf areabased foliar RGR for ssp. tridentata (Tables 2 and 3). After
the freezing treatment was imposed, growth for both
subspecies nearly ceased. The freezing treatment was the
only treatment that significantly affected plant biomass
(Tables 2 and 3). Both root and shoot biomass were
reduced by approximately 40% in plants exposed to the
freezing treatment. Root:shoot were unaffected by either
low moisture or freezing. Furthermore, following the
freezing treatment, differences in growth rates between
the moisture treatments became apparent, such that
plants grown under low moisture had 30% slower growth
rates than those under high moisture (Tables 2 and 3). SLA
was also different between the subspecies and the
moisture treatments, where ssp. tridentata had 8% lower
SLA (thicker leaves) than ssp. vaseyana and plants in the
low-moisture treatment had 10% lower SLA than plants in
the high moisture treatment (Tables 2 and 3).
Photosynthetic gas exchange exhibited varying responsiveness to the treatments. Values of Amax were reduced
by both low moisture and freezing for both subspecies
(Fig. 3, Table 3). In contrast, values of gs were similar
across the moisture treatments, but differed across subspecies and following the freezing treatments, where ssp.
tridentata had lower rates than ssp. vaseyana (Fig. 3C, D,
Table 3). WUE was not affected by the treatments for
either subspecies (Tables 2 and 3).
The two measures of PSII quantum yield indicate that
both subspecies were under moderate levels of stress
when exposed to the drought and freezing treatments.
Whereas FPSII declined under both low moisture and
after the freezing treatment, Fv/Fm declined only with low
moisture (Fig. 4, Table 3). For ssp. tridentata, freezing
plants grown under the low-moisture treatment caused
a decline in FPSII, but low moisture alone did not (Fig. 4).
For ssp. vaseyana, both the freezing treatment and the
low-moisture treatment led to a decline in FPSII, both
alone and in combination (Fig. 4). Values of Fv/Fm
declined slightly and significantly only for ssp. vaseyana
under the freezing low-moisture treatment (Fig. 4).
Discussion
Morphological and physiological differences associated
with drought tolerance between seedlings of ssp.
vaseyana and ssp. tridentata in this comparative greenhouse study were consistent with previous studies of
211
Table 2. Average values (1 SE) for several measures of plant size and growth. See Table 3 for statistics.
Subspecies vaseyana
Variable
RGR (prefreeze)
(cm2 cm22 day21)
RGR (postfreeze)
(cm2 cm22 day21)
SLA (cm2 g21)
Final root
biomass (g)
Final shoot
biomass (g)
Root:shoot
WUE (mmol mmol21)
Subspecies tridentata
Low
moisture
Low
moisture freeze
High
moisture
High
moisture freeze
Low
moisture
Low
moisture freeze
High
moisture
High
moisture freeze
0.004 (0.003)
—
0.006 (0.004)
—
0.007 (0.004)
—
0.009 (0.002)
—
0.004 (0.001)
20.01 (0.009)
159.5 (12.1)
0.03 (0.004)
—
0.009 (0.002)
178.0 (9.3)
0.05 (0.02)
—
0.008 (0.007)
0.005 (0.001)
3.9 (0.6)
5.3 (4.2)
1.8 (0.3)
18.2 (8.1)
0.02 (0.005)
0.002 (0.005)
0.02 (0.002)
0.007 (0.002)
0.03 (0.007)
0.00 (0.007)
0.2 (0.02)
148.1 (8.7)
0.03 (0.002)
—
0.007 (0.002)
167.8 (17.9)
0.01 (0.006)
—
0.02 (0.006)
0.013 (0.005)
0.008 (0.003)
0.02 (0.006)
0.004 (0.001)
0.03 (0.01)
0.03 (0.01)
3.2 (0.8)
16.7 (4.3)
2.4 (0.7)
3.0 (8.7)
2.1 (0.6)
7.3 (3.8)
2.4 (1.1)
20.5 (4.1)
1.9 (0.5)
15.7 (13.4)
1.9 (0.4)
1.8 (0.2)
mature individuals (Barker and McKell 1986, Frank et al.
1986, McArthur and Welch 1982, Shultz 1986a, Welch
and McArthur 1986). In particular, ssp. tridentata displayed a suite of traits that favored both higher water
conservation and early-season water use than ssp.
vaseyana, which would be important adaptive features
in the lower elevation (relatively more xeric) habitats of
ssp. tridentata. The thicker leaves (i.e. lower SLA) and
lower gs of ssp. tridentata reduce water loss and are
expected to promote drought tolerance (Lambers et al.
1998). This suite of traits that would promote relatively
greater drought tolerance for mature ssp. tridentata (Frank
et al. 1986, Shultz 1986a) has to our knowledge not been
previously observed for seedlings.
Rapid growth capacity is another adaptive feature of
drought-tolerant plants that enables them to take advantage of early-season soil moisture. We observed higher
aboveground RGR for ssp. tridentata than for ssp.
vaseyana. Similar results between these subspecies have
been previously observed for both seedlings (Booth et al.
Table 3. Statistical results of generalized linear model comparing the effects of subspecies, long-term moisture treatment and episodic freezing
treatment on plant size, growth and physiology. Analyses on RGR prior to freezing (RGR prefreeze) compare growth among seedlings under the moisture
treatments that did not undergo freezing. RGR (postfreeze) compares growth of seedlings in all treatments during the period following the imposition of
freezing. Those analyses that are statistically significant at the a ¼ 0.05 level are shown in bold.
Variable
Plant size and growth
Final leaf area (cm2)
RGR (prefreeze) (cm2 cm22 day21)
RGR (postfreeze) (cm2 cm22 day21)
SLA (cm2 g21)
Final root biomass (g)
Final shoot biomass (g)
Root:shoot
Gas exchange
Amax (mmol m22 s21)
gs (mmol m22 s21)
WUE (mmol mmol21)
Quantum efficiency
FPSII
Fv/Fm
212
Ssp freeze
Ssp moisture freeze
P
F
P
F
P
2.96
0.06
1.42
0.59
0.24
1.29
0.09
0.09
0.80
0.24
0.45
0.62
0.26
0.76
5.56
—
2.43
—
0.08
0.41
3.20
0.03
—
0.13
—
0.78
0.53
0.08
4.12
—
3.89
—
0.23
0.05
0.77
0.05
—
0.05
—
0.63
0.82
0.39
0.001
0.02
0.30
0.99
0.89
0.59
1.34
3.37
0.19
0.26
0.08
0.67
0.001
7.79
0.05
0.97
0.01
0.83
0.22
0.25
0.65
0.62
0.68
0.76
0.42
0.39
1.89
1.88
0.19
0.18
Ssp moisture
Subspecies
Moisture
Freeze
F
P
F
P
F
P
F
4.79
6.87
2.88
9.33
0.27
3.16
2.70
0.04
0.01
0.10
0.01
0.60
0.08
0.11
17.87
3.26
8.61
16.81
2.12
3.03
0.22
<0.001
0.08
0.01
0.001
0.16
0.09
0.64
15.00
—
8.42
—
5.38
4.55
2.31
<0.001
—
0.01
—
0.02
0.04
0.14
0.57
12.84
0.04
0.46
0.001
0.85
4.92
0.01
0.63
0.05
0.91
0.43
11.28
10.68
0.08
0.003
0.003
0.78
3.88
3.53
0.06
0.07
18.95
5.28
<0.001
0.03
18.19
2.58
<0.001
0.11
Physiol. Plant. 130, 2007
A
6
B
ssp. tridentata
b
6
4
4
b
2
2
ab
a
a
a
a
Leaf area
(cm2/plant)
Leaf area
(cm2/plant)
ssp. vaseyana
Low moist x freeze
Low moist
High moist x freeze
High moist
a
0
0
Fig. 2. Average total leaf area per plant (1 SE) under each of the treatments. For each subspecies, bars sharing a common letter are not significantly
different at a ¼ 0.05 according to Tukey post hoc pairwise comparisons. n ¼ 5 per treatment. See Table 3 for statistics.
1990) and adults (Barker and McKell 1986, Frank et al.
1986, McArthur and Welch 1982, Welch and McArthur
1986). Our observed subspecies-level differences in RGR
became less pronounced later in the study period as
compared with measurements earlier in the study. The
differences we observed between the sets of growth
measurements are likely because of the fact that at the end
of the experiment when the last set of leaf area measures
were made, seedlings of both subspecies were exhibiting diminished growth that is typical of plants at the end of
the growing season. This conclusion is supported by previous work on A. tridentata seedling growth rates, which
showed that 15 weeks after seed sowing (which would
be comparable to the end of our experiment), growth of
both these subspecies was in decline (Booth et al. 1990).
B
Low moisture x freeze
Low moisture
High moisture x freeze
High moisture
20
15
b
ab
a
25
20
15
ab
a
10
a
a
10
a
5
1.2
5
D
C
1.2
b
1.0
1.0
0.8
b
0.8
b
0.6
a
0.4
0.2
0.0
0.6
a
a
a
a
0.4
gs (mmol m–2 s–1)
gs (mmol m–2 s–1)
ssp. tridentata
ssp. vaseyana
A
Amax (µmol m–2 s–1)
Amax (µmol m–2 s–1)
25
While another growth strategy for plants in xeric environments is to devote early-season resources to belowground
growth, we did not observe subspecies differences in total
root biomass in our study.
In spite of the differences observed between the
subspecies and the relatively greater drought-tolerating
characteristics exhibited by ssp. tridentata, both subspecies responded similarly to the low-moisture treatment. That is, both subspecies showed a similar level of
overall physiological tolerance to low moisture, even
though they had lower growth rates than in the high
moisture treatment. Drought tolerance is probably more
essential for seedlings than mature plants, due in part
to small total root area and rooting depth. Mature A. tridentata can have roots to 3 m or deeper, where mid- and
0.2
0.0
Fig. 3. Average maximum photosynthetic assimilation (Amax) and stomatal conductance to water vapor (gs) (1 SE) for each subspecies under each
treatment. Bars sharing a common letter within each panel are not significantly different at a ¼ 0.05 according to Tukey post hoc pairwise comparisons.
n ¼ 5 per treatment. See Table 3 for statistics.
Physiol. Plant. 130, 2007
213
0.3
A
ssp. vaseyana
ssp. tridentata
b
b
b
0.3
ab
a
0.2
0.2
a
Φ PSII
Φ PSII
Low moist x freeze
Low moist
High moist x freeze
High moist
B
a
a
0.9
0.9
a
b
0.8
0.7
b
b
a
a
a
0.8
Fv /Fm
Fv /Fm
a
0.7
Fig. 4. The result of the freezing treatment on chlorophyll a fluorescence from PSII for seedlings of each subspecies grown under each of the moisture
treatments. Average ( 1 SE) dark-adapted quantum efficiency (Fv/Fm) and light-adapted quantum efficiency (FPSII). Both measures are dimensionless.
n ¼ 5 per treatment. See Table 3 and text for statistics.
late-season soil moisture remains higher than that found
near the surface (Campbell and Harris 1977). Seedlings of
both subspecies that have yet to produce such deep roots
likely face lower soil water availability earlier in the
growing season than mature plants. The similar root:shoot
for both subspecies suggests that their allocation patterns
are similar at the seedling stage.
The single, episodic freezing treatment produced
relatively greater impacts on the seedlings than did the
long-term soil moisture treatment. For plants exposed to
the freezing treatment, growth essentially stopped, which
was indicated by the smaller final plant sizes relative to the
control plants. The decrease in light-adapted PSII quantum
yield for both subspecies following the single freezing
event also indicates that photosynthetic tissues were
somewhat affected by freezing. These reductions are
similar to those reported at similar temperatures for ssp.
tridentata seedlings under controlled environments (Loik
and Redar 2003) but were more sensitive to freezing than
for mature ssp. vaseyana measured in situ (Loik et al.
2004b). This is consistent with the general notion that
seedlings of many species (including A. tridentata in this
case) are more susceptible to freezing stress than are
mature individuals. Not only can freezing temperatures
damage plant tissues, they can also dehydrate plant tissues
and cause stress in similar manners as drought, such as
increasing xylem cavitation rates (Sakai and Larcher 1987,
Lambers et al. 1998), or by freeze distillation of water from
214
photosynthetic mesophyll cells (Loik and Nobel 1991,
1993). Molecular studies have shown that freezing
and drought can induce expression of the same genes,
in part because plants produce similar signals in response
to the osmotic stresses created by freezing and drought
(Kacperska 2004, Yakashima and Yamaguchi-Shinozaki
2006). Both Amax and gs were reduced in plants that experienced freezing, suggesting such tissue dehydration
occurred and resulted in lasting effects on stomatal function, photosynthetic carbon assimilation and leaf production. Diurnal measurements (data not shown) showed
similar patterns of reduced Amax and gs in plants in lowmoisture and/or freezing treatments.
Overall, the freezing treatment affected the subspecies
similarly. However, leaf area, Fv/Fm and gs of ssp.
vaseyana were reduced more by freezing for plants
grown under low than high moisture. Therefore, its
susceptibility to freezing stress was increased by the lowmoisture treatment. Similar results have been found in
seedlings of other plant species, where drought can
exacerbate the effects of freezing (Bruelheide and
Heinemeyer 2002). In contrast, certain other species are
more vulnerable to freezing damage when well hydrated
(Loik and Nobel 1991, 1993).
In conclusion, our hypothesis was not supported: both
subspecies were similarly affected by both the long-term
drought and single, episodic freezing treatments. However, both subspecies showed some degree of tolerance to
Physiol. Plant. 130, 2007
low moisture (a condition both likely face while seedlings), yet they were relatively more sensitive to freezing
and to the combined freezing low-moisture treatment.
The implications of these results are important for
understanding plant community shifts under climate
change. Whereas climate models predict a warming
trend throughout much of the Great Basin region (Field
et al. 1999, Watson et al. 1996), the occurrence of growing season episodic freezing events is likely to continue
(Loik et al. 2004b). Also, climate model scenarios of
future precipitation patterns and soil water availability for
plants are highly uncertain (Loik et al. 2004a). Because
freezing temperatures are considered more important in
predicting plant distributions than average temperatures
(Bruelheide and Heinemeyer 2002, Luo and Mooney
1999, Nardini et al. 1998), our results show the importance of freezing effects on both subspecies of A. tridentata, which may become particularly exacerbated
under lower moisture availability. Furthermore, the tolerance of mature individuals to particular environmental
conditions does not predict the tolerance of seedlings to
those conditions; while soil moisture is considered
important in determining the range at the species- and
subspecies-level for mature A. tridentata, episodic freezing events may be relatively more important for seedlings.
Acknowledgements – We thank R. Fernández and D. Tissue
for assistance in designing the moisture controlling method.
We would also like to thank J. Velzy and L. Locatelli for
greenhouse facilities and management and for assistance with
setup and design of the experiment. Western Native Seed of
Coaldale, CO, assisted with collecting seeds.
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