Variations in precipitation and atmospheric N deposition affect water and N
availability in desert and thus may have significant effects on desert
ecosystems. Haloxylon ammodendron is a dominant plant in Asian desert, and addressing its
physiological acclimatization to the changes in precipitation and N
deposition can provide insight into how desert plants adapt to extreme
environments by physiological adjustment. Carbon isotope ratio (δ13C) in plants has been suggested as a sensitive long-term indicator
of physiological acclimatization. Therefore, this study evaluated the effect
of precipitation change and increasing atmospheric N deposition on δ13C of H. ammodendron. Furthermore, H. ammodendron is a C4 plant; whether its δ13C can indicate water use efficiency (WUE) has not been addressed. In
the present study, we designed a field experiment with a completely
randomized factorial combination of N and water and measured δ13C and gas exchange of H. ammodendron. Then we calculated the degree of
bundle-sheath leakiness (φ) and WUE of the assimilating branches of
H. ammodendron. δ13C and φ remained stable under N and water supply,
while N addition, water addition and their interaction affected gas exchange
and WUE in H. ammodendron. In addition, δ13C had no correlation with WUE.
These results were associated with the irrelevance between δ13C
and the ratio of intercellular to ambient CO2 concentration
(ci/ca), which might be caused by a special value (0.37) of the
degree of bundle-sheath leakiness (φ) or a lower activity of
carbonic anhydrase (CA) of H. ammodendron. In conclusion, δ13C of H. ammodendron is not
sensitive to global change in precipitation and atmospheric N deposition
and cannot be used for indicating its WUE.
Introduction
Recently, global precipitation pattern has changed significantly (Frank et
al., 2015; Knapp et al., 2015), and atmospheric N deposition has continued
to rise (Galloway et al., 2004; Liu et al., 2013; Song et al., 2017).
Previous researchers have suggested that arid ecosystems are most sensitive
to climate change (Reynolds et al., 2007; Huang et al., 2016), while global
change in precipitation and atmospheric N deposition has an important impact
on water and N availability in desert (Huang et al., 2018). Thus, these
changes may have significant effects on desert ecosystems. Haloxylon ammodendron is a dominant
species in desert regions, especially in Asia. Studying the physiological
responses of H. ammodendron to global change can provide insight into how desert plants
adapt to extreme environments by physiological adjustment. Carbon isotope ratio
(δ13C) in plants depends on the ratio of intercellular to
ambient CO2 concentration (ci/ca), which reflects the balance
between inward CO2 diffusion rate, regulated by stomatal conductance
(gs), and CO2-assimilating rate (A) (Farquhar and Richards, 1984)
and has been suggested as a sensitive long-term indicator of physiological
acclimatization (Battipaglia et al., 2013; Cernusak et al., 2013; Tranan and
Schubertt, 2016; Wang and Feng, 2012). Therefore, investigating the
variations in δ13C of H. ammodendron under water and nitrogen addition can
enhance understanding of physiological responses of desert plants to future
changes in precipitation and atmospheric N deposition.
A large quantity of works have been devoted to the relationships between
C3 plant δ13C and water availability or precipitation
(e.g., Diefendorf et al., 2010; Kohn, 2010; Liu et al., 2005; Ma et al.,
2012; Serret et al., 2018; Stewart et al., 1995; Wang et al., 2005, 2008)
and nitrogen availability (e.g., Cernusak et al., 2007; Li et al., 2016;
Sparks and Ehleringer, 1997; Yao et al., 2011; Zhang et al., 2015). However,
a relatively small amount of research has focused on the responses of
C4 plant δ13C to water availability or precipitation
(Ellsworth et al., 2017; Liu et al., 2005; Rao et al., 2017; Wang et al.,
2006) and nitrogen availability (Ma et al., 2016; Schmidt et al., 1993). For
C4 plants, δ13C is controlled by both the ci/ca
ratio and the degree of bundle-sheath leakiness (φ), the proportion
of CO2 produced within bundle-sheath cells from C4 acids that
leaks back to mesophyll cells (Ellsworth and Cousins, 2016; Ellsworth et
al., 2017; Farquhar, 1983). Thus, the responses of C4 plant δ13C to water and N availability are also affected by φ.
Genetic factors control φ values, which causes the interspecific
differences in δ13C, even the responses of plant δ13C to water and N availability (Gresset et al., 2014). On the other
hand, enzymatic activity of carbonic anhydrase (CA) may influence δ13C in C4 plants (Cousins et al., 2006). CA is an enzyme that
catalyzes the hydration of CO2 in mesophyll cells to form bicarbonate
(HCO3-). Previous studies showed that CA activity in most C4
plants is usually low, just sufficient to support photosynthesis (Cousins et
al., 2006; Gillon and Yakir, 2000, 2001; Hatch and Burnell, 1990). H. ammodendron is a
typical C4 plant. How its δ13C responds to water and N
availability has never been addressed.
Foliar δ13C in C3 plants has been considered as a useful
indicator of intrinsic water use efficiency (WUE) (Farquhar, 1983). However,
although some studies have suggested that δ13C of C4
plants could also indicate its WUE (Henderson et al., 1992; Wang et al.,
2005; Cernusak et al., 2013; Ellsworth and Cousins, 2016), this statement is
still controversial. The relationship between δ13C and WUE is
based on the links between ci/ca ratio and δ13C and
between ci/ca ratio and WUE (Ehleringer and Cerling, 1995). For
C3 plants, δ13C always decreases with an increase in
ci/ca ratio; but for C4 plants, the correlation between
δ13C and ci/ca ratio depends on the φ value
(Cernusak et al., 2013) and CA activity (Cousins et al., 2006). As mentioned
above, φ value is under genetic control, and the CA activity
changes across species (Cousins et al., 2006; Gillon and Yakir, 2000, 2001;
Hatch and Burnell, 1990); thus, the correlation between δ13C
and ci/ca ratio, as well as the relationship between WUE and
δ13C, shows interspecific difference. Whether δ13C
of H. ammodendron indicates WUE has never been evaluated.
In this study, we designed an experiment with multiple water and nitrogen
supplies in the southern Gurbantünggüt Desert in Xinjiang Uygur Autonomous
Region, China. We measured the δ13C, gas exchange and WUE of
the assimilating branches of H. ammodendron. We had two objectives. One objective was to
evaluate the response of the dominant plant of Asian desert to future
changes in precipitation and atmospheric N deposition by revealing the
effects of water and N supply on δ13C of H. ammodendron. The other was to
explore the availability of δ13C as the indicator of water
use efficiency in H. ammodendron.
Materials and methodsDefinitions and basic equations
Stable carbon isotopic ratio (δ13C) of natural materials is
expressed as
δ13C(‰)=(13C/12C)sample(13C/12C)standard-1×1000,
where (13C /12C)sample and (13C /12C)standard
are the 13C /12C ratio of the sample and of the Pee Dee Belemnite
(PDB) standard, respectively. Farquhar (1983) proposed the pattern of carbon
isotopic discrimination (Δ) in C4 plants:
Δ=δ13Cair-δ13Cplant1+δ13Cplant/1000≈δ13Cair-δ13Cplant=a+b4+φb-s-acica,
where δ13Cplant and δ13Cair are the
δ13C values of plants and CO2 in the ambient air,
respectively. The parameter a (= 4.4 ‰; Craig, 1954)
is the carbon isotopic fractionation in the diffusion of CO2 into
internal leaves; b4 (=-5.9 ‰; O'Leary, 1984) is
the combined carbon isotopic fractionations occurring in the processes of
gaseous CO2 dissolution, hydration–dehydration reactions of CO2
and HCO3- in mesophyll cells, and HCO3- carboxylation
by PEP (phosphoenolpyruvate) carboxylase; s (= 1.8 ‰;
O'Leary, 1984) is the carbon isotopic fractionation during diffusion of
CO2 out of the bundle-sheath cells; and b (= 27 ‰; Farquhar and Richards, 1984) is the carbon isotopic fractionation of
CO2 carboxylation by RuBP (ribulose-1,5-bisphosphate) carboxylase. The
variable φ is the proportion of CO2 production within bundle-sheath cells from C4 acids that leaks back to mesophyll cells, and
ci/ca is the ratio of intercellular to ambient CO2
concentration. Eq. (2) can be transformed into the following format:
δ13Cplant=-b4+φb-s-acica+δ13Cair-a.
According to Eq. (3), if the coefficient [b4+φ (b-s) -a] is greater than 0, δ13C decreases with increasing
ci/ca; if this coefficient is lower than 0, δ13C
increases with increasing ci/ca.
Water use efficiency (WUE) is defined as the amount of assimilated carbon
dioxide by plants under the consumption per unit of water. There are two
characteristics of WUE, instantaneous WUE (ins-WUE) and intrinsic WUE
(int-WUE). Ins-WUE can be calculated by
ins-WUE=A/E=(ca-ci)/1.6v=ca(1-ci/ca)/1.6v,
where A is photosynthetic rate, E is transpiration rate and v is calculated
by
v=(ei-ea)/p,
where ei and ea are the water vapor pressure inside and outside
the leaves, and p is the atmospheric pressure.
The definition of int-WUE is
int-WUE=A/gs=(ca-ci)/1.6=ca(1-ci/ca)/1.6,
where gs is stomatal conductance.
Study site and species
This experiment was conducted at the Fukang Station of Desert Ecology,
Chinese Academy of Sciences, on the southern edge of the Gurbantünggüt
Desert (44∘26′ N, 87∘54′ E) in
northwestern China. The altitude of the study site is 436.8 m above average
sea level (a.s.l.). It is a typical continental arid, temperate climate,
with a hot summer and cold winter in the area. The mean annual temperature
is 7.1 ∘C, and the mean annual precipitation is 215.6 mm,
with a potential evaporation of about 2000 mm. The mean annual temperature
and the annual rainfall amount in the sampling year are 10.23 ∘C and 122.7 mm (Cui, 2018). The soil type is grey desert
soils (Chinese classification) with aeolian sands on the surface (0–100 cm).
The percentages of clay (< 0.005 mm), silt (0.005–0.063 mm), fine
sand (0.063–0.25 mm) and medium sand (0.25–0.5 mm) range from 1.63 %–1.76 %,
13.79 %–14.15 %, 55.91 %–56.21 % and 20.65 %–23.23 %, respectively (Chen et
al., 2007). The soil is highly alkaline (pH = 9.55 ± 0.14) with low
fertility. The vegetation is dominated by Haloxylon ammodendron and Haloxylon persicum with about 30 % coverage.
Herbs include ephemerals, annuals and small perennials, with a cover of ca.
40 % (Fan et al., 2013). Although the coverage of the two Haloxylon species is a
little lower than that of herbs, the biomass of the former is much larger
than that of the latter, because Haloxylon plants are shrubs with an average height of
1.5 m whereas the latter are very low herbaceous plants. Biological soil
crusts are distributed widely on the soil between the herbs and Haloxylon, with
almost 40 % coverage (Zhang et al., 2007).
The present study focused on Haloxylon ammodendron because it is the dominant species in Asian
desert. H. ammodendron is a species of Chenopodiaceae, which is a xerophytic and
halophytic woody plant (Cui et al., 2017). The leaves of H. ammodendron have been
completely degraded due to the extreme drought, and the assimilation
branches, which are the glossy green branches (Fig. S1), perform the same
functions as the leaves. Due to its drought tolerance, H. ammodendron is widely
distributed in desert areas.
Experimental design
A field experiment with a completely randomized factorial combination of
water and nitrogen has been conducted from 2014 to 2017. We designed two
water addition levels (0, 60 mm yr-1; W0, W1) based on the
prediction that precipitation will increase by 30 % in northern China in
the next 30 years (Liu et al., 2010), and three levels of N addition (0, 30,
60 kg N ha-1 yr-1; N0, N1 and N2), because N
deposition has reached 35.4 kg N ha-1 yr-1
in the nearby city Ürümqi (Cui et al., 2017) and will double by 2050
relative to the early 1990s (Galloway et al., 2008). Therefore, there were
six treatments (W0N0, W0N1, W0N2, W1N0, W1N1 and W1N2) in this experiment. Four
replicates of each treatment were set, making a total of 24 plots with a
size of 10 m × 10 m. A small sub-plot with a size of 1.5 m × 1.5 m was set in each plot. A well-grown H. ammodendron was enclosed in the
center of the sub-plot. The average height and coverage of an individual H. ammodendron
were 1.5 m and 1.9 m2, respectively, and did not vary significantly
across the plots. The type of nitrogen used in the present study is
NH4NO3. To simulate natural water and N inputs, the treatments
were applied in equal amounts, 12 times, once a week in April, July and
September, as 5 mm m-2 of water and 2.5 or 5 kg N ha-1 each week (Cui et al., 2017). Usually, water addition
was with a sprinkler kettle, irrigating over the canopy of H. ammodendron.
Measurements of gas exchange and WUE
As mentioned above, the main assimilating organ of H. ammodendron is the assimilation
branches. Thus, we conducted gas exchange measurements on the assimilation
branches of the H. ammodendron grown in the sub-plots by a LI-6400 portable photosynthesis
system. The measurements were conducted on 27–29 June 2016, which is the
main growing season of H. ammodendron. It may be most appropriate to take measurements
during this period, and the results of the measurements are therefore more
representative. Previous studies have also usually conducted this
measurement during the growing season (Nyongesah and Wang, 2013; Cui, 2018;
Gong et al., 2019). The ins-WUE was calculated based on these measured gas
exchange traits by Eq. (4) and int-WUE by Eq. (6). At each plot, the
top assimilating branches of a mature individual were selected randomly for
the measurement of gas exchange, which includes photosynthetic rate (A),
stomatal conductance (gs), transpiration rate (E), the ambient CO2
concentration (ca) and the intercellular CO2 concentration
(ci). Before the measurement of gas exchange, it takes about 5 s to
stabilize after the assimilating branches were inserted in the cuvette. We
repeated 10 times on the same assimilating branches for each measurement.
Our measurements were carried out under the conditions of a standard 450 mmol mol-1 CO2 concentration at a flow rate of 500 mmol s-1 above saturation in photo flux density of 1600 mmol m-2 s-1. The temperature of the assimilating branches varied from 29.5 to 30.5 ∘C during the entire period of gas exchange measurements.
Sample collection
Sample collection was conducted in 20 July, during the addition of water
and nitrogen. Considering that there is a considerable difference in δ13C between buds and young and matured leaves, we collected the mature
assimilating branches of H. ammodendron for the δ13C measurements. All H. ammodendron
individuals grown in plots (10 m × 10 m) were sampled. Eight pieces
of the mature assimilating branches (15–20 cm long) were collected from each
individual; two pieces of assimilating branches were collected at each of
the four cardinal directions from the positions of full irradiance. All
assimilating branches from the same plot were combined into one sample.
After the samples were collected, they were immediately divided into two
parts randomly and taken back to the laboratory at Fukang Station. The first
part was used to determine the chlorophyll content. The second part was
immediately inactivated in a 105 ∘C oven in the laboratory at
Fukang Station and then brought back to Beijing in a ziplock bag. The time
interval between sample collection and inactivation is very short. After
inactivation, the carbon exchange of the assimilating branches stops, so the
isotope composition of the samples will not change anymore. All plant
samples of the second part were air-dried immediately in the laboratory in
Beijing. Then the samples were ground into a fine powder using a steel ball
mixer mill MM200 (Retsch GmbH, Haan, Germany) for the measurements of
δ13C and N contents.
Measurements of plant δ13C, plant N and chlorophyll
contents
The δ13C and N measurements were performed on a DeltaPlus
XP mass spectrometer (Thermo Scientific, Bremen, Germany) coupled with an
automated elemental analyzer (Flash EA1112, CE Instruments, Wigan, UK) in
continuous-flow mode, at the Stable Isotope Laboratory of the College of
Resources and Environmental Sciences, China Agricultural University. The
carbon isotopic ratios were reported in the delta notation relative to the
V-PDB standard. For this measurement, we obtained standard deviations lower
than 0.15 ‰ for δ13C among replicate
measurements of the same sample. And standard deviations for the N
measurements were 0.1 %.
The chlorophyll contents of all samples were determined immediately when the
samples were taken back in the laboratory at Fukang Station. The samples were
first extracted by 95 % ethyl alcohol (0.5 g sample to 25 mL ethyl
alcohol), and then the absorbency was measured under the wavelengths 665
and 649 mm by the spectrophotometer. The content of chlorophyll a and b was
calculated by the following equations:
7Chlorophylla(mg/L)=13.95×OD665-6.88×OD649,8Chlorophyllb(mg/L)=24.96×OD649-7.32×OD665,
where OD665 and OD649 are the absorbency under the wave lengths of 665 and
649 mm, respectively.
Calculation of the degree of bundle-sheath leakiness
The degree of bundle-sheath leakiness (φ) was calculated by the
transformation of Eq. (2):
φ=(δ13Cair-δ13Cplant)/(1+δ13Cplant/1000)-aci/ca+a-b4/b-s.
In this equation, parameters a, b4, b and s are constant, while δ13Cplant and ci/ca are the measured values of our
samples. We did not measure the δ13Cair at our study
site, so we had to use an approximation of the δ13Cair to do this ϕ calculation. The approximated value we used is
-9.77 ‰, which was measured at Donglingshan, Beijing, north China, in September 2019. The two sites should have
similar δ13Cair because the two sites are located in
countryside with less human activity and have a similar distance from the
nearest city. The straight line distances between Donglingshan and
the city center of Beijing as well as between our study site and Ürümqi city are
about 90 km. In addition, since the δ13Cair has large
diurnal and seasonal variations, we used the published range of δ13Cair from May to July in Shangdianzi, China (data come from
Global Monitoring Laboratory, Earth System Research Laboratories,
https://www.esrl.noaa.gov/gmd, last access: 21 March 2021) to calculate the minimum and maximum δ13Cair of the time period, which is the main growing season for
H. ammodendron. Finally, the δ13Cair used in the calculation ranged
from -10.52 ‰ to -9.01 ‰ with an
average of -9.77 ‰.
Statistical analysis
Statistical analyses were conducted using SPSS software (SPSS for Windows,
Version 20.0, Chicago, IL, United States). One-way analysis of variance
(ANOVA) and two-way analysis of variance (ANOVA) were used to compare the
difference of δ13C and other physiological traits between each
treatment. Pearson analysis was used to determine the correlation among
δ13C, WUE and ci/ca in H. ammodendron.
ResultsPlant δ13C under water and nitrogen addition
The δ13C of the assimilating branches of H. ammodendron in the six treatments
W0N0, W0N1, W0N2, W1N0, W1N1 and W1N2 was -14.18± 0.19 ‰, -14.71± 0.35 ‰, -14.45± 0.18 ‰, -14.67± 0.40 ‰, -14.65± 0.38 ‰ and -14.344± 0.29 ‰. One-way ANOVA
showed no significant variation in δ13C across treatments (p= 0.79, Fig. 1). Two-way ANOVA suggested that δ13C
was not affected by water addition (p= 0.68), N addition (p= 0.61) or
their interaction (p= 0.56, Table 1).
The p values of all measured and calculated indexes in plants under
two-way ANOVA analysis of water (W) and nitrogen (N) additions.
Note. φmin, φave and φmax
represent the φ values calculated from the minimum, average and
maximum δ13Cair .*, ** and *** indicate a significant influence. W ⋅ N represents the interaction between water addition and N addition.
The δ13C of assimilating branches of Haloxylon ammodendron under water (W)
and nitrogen (N) additions. The spot represents the mean value of four
replicates with error bars denoting the standard error (SE).
Gas exchange and WUE under water and nitrogen addition
Photosynthetic rate (A), stomatal conductance (gs), transpiration rate
(E) and ci/ca ranged from 12.11 to 39.35 µmol CO2 m-2 s-1, from 0.09 to 0.31 mol H2O m-2 s-1, from 2.87 to 8.49 mmol H2O m-2 s-1 and
from 0.11 to 0.57, respectively. One-way ANOVA showed significant
changes in leaf gas exchange across the six treatments (p< 0.01 for
A, gs, E and ci/ca, Fig. 2). Two-way ANOVA suggested
that water addition had exerted an effect on ci/ca (p< 0.01), that N additions influenced A (p< 0.01) and ci/ca (p= 0.009), and that the interaction between water and N supply played
a role in gs (p< 0.001), E (p< 0.001) and
ci/ca (p< 0.001, Table 1).
Variations in photosynthetic rate (a), stomatal conductance (b),
water use efficiency (c) and ci/ca(d) across water (W) and
nitrogen (N) additions. The spot represents the mean value of four
replicates with error bars denoting the standard error (SE).
Instantaneous WUE (ins-WUE) and intrinsic WUE (int-WUE) ranged from 3.09 µmol CO2/ mmol H2O to 8.49 µmol CO2/ mmol H2O and from 93.64 µmol CO2/ mol H2O to 208.47 µmol CO2/ mmol H2O, respectively. One-way ANOVA showed
significant changes in these two indexes across these treatments (both p< 0.001, Fig. 3). Two-way ANOVA suggested that water
addition, N addition and their interaction all have a significant effect on
these two indexes (all p< 0.05, Table 1).
Variations in ins-WUE (a) and int-WUE (b) across water (W) and
nitrogen (N) additions. The spot represents the mean value of four
replicates with error bars denoting the standard error (SE).
Correlations among δ13C, WUE and ci/ca ratio
In order to test whether δ13C in H. ammodendron can indicate WUE, the
relationships among δ13C, ins-WUE, int-WUE and ci/ca
ratio were revealed in this study. Our results showed no correlation between
δ13C and ins-WUE (p= 0.23, Fig. 4a), between δ13C and int-WUE (p= 0.23, Fig. 4c), or between δ13C
and ci/ca ratio (p= 0.18, Fig. 4e). However, there was a
negative correlation between ins-WUE and ci/ca ratio (p< 0.001, Fig. 4b) and between int-WUE and ci/ca ratio (p< 0.001, Fig. 4d).
Correlations of ins-WUE vs. δ13C (a), ins-WUE vs.
ci/ca(b), int-WUE vs. δ13C (c), int-WUE vs.
ci/ca(d) and δ13C vs. ci/ca(e) of
assimilating branches of Haloxylon ammodendron.
The degree of bundle-sheath leakiness under water and nitrogen addition
The φ value calculated from the minimum δ13Cair
ranged from 0.16 to 0.50 with a mean value of 0.35; the φ value
calculated from the maximum δ13Cair ranged from 0.44 to
0.70 with a mean value of 0.55, and the φ value calculated from the
average δ13Cair ranged from 0.32 to 0.59 with a mean value
of 0.45. One-way ANOVA showed no significant variation in φ calculated from the minimum, average and maximum δ13Cair
across treatments (p= 0.60 for the φ calculated from the minimum
δ13Cair, p= 0.77 for the φ calculated from the
average δ13Cair and p= 0.90 for the φ
calculated from the maximum δ13Cair, Fig. 5). Two-way
ANOVA suggested that φ was not affected by water addition
(p= 0.46 for the φ calculated from the minimum δ13Cair, p= 0.64 for the φ calculated from the average
δ13Cair and p= 0.98 for the φ calculated
from the maximum δ13Cair), N addition (p= 0.65 for the
φ calculated from the minimum δ13Cair, p= 0.60
for the φ calculated from the average δ13Cair
and p= 0.55 for the φ calculated from the maximum δ13Cair) or their interaction (p= 0.30 for the φ
calculated from the minimum δ13Cair, p= 0.52 for the
φ calculated from the average δ13Cair and p= 0.87 for the φ calculated from the maximum δ13Cair, Table 1).
Variations in φ calculated by Eq. (9) using the minimum (Min),
average (Ave) and maximum (Max) values of δ13Cair across
water (W) and nitrogen (N) additions. The box represents the mean value of
four replicates with error bars denoting the standard error (SE).
Discussion
The δ13C of the assimilating branches in H. ammodendron did not change across
treatments (Fig. 1, Table 1), suggesting that neither water addition nor
nitrogen addition influenced the δ13C of H. ammodendron. Previous studies
also reported no significant relationship between δ13C of
C4 plant and water availability (Swap et al., 2004; Wang et al., 2008)
and between δ13C of C4 plant and nitrogen availability
(Yao et al., 2011; Yang et al., 2017).
In general, the effects of water availability and nitrogen availability on
δ13C are dependent on ci/ca ratio, which reflects the
balance between stomatal conductance (gs) and photosynthetic rate (A)
(Farquhar and Richards, 1984). Stomatal conductance (gs) usually
increases with increasing water availability under water addition. Although
two-way ANOVA suggested that water addition had no effect on both A
and gs (Table 1), one-way ANOVA showed that gs was
higher in W1N0 than that in W0N0 (Fig. 2b), indicating that water addition
had a positive effect on gs under ambient N conditions. Increasing
gs under water supply will lead to the rise of intercellular CO2
because of the decrease in diffusional resistance to CO2. As a
result, ci/ca ratio was observed to increase with increasing
moisture (Fig. 2d, Table 1). However, δ13C remained stable
under water addition (Fig. 1, Table 1). Thus, ci/ca ratio could
not explain the observed response of δ13C to water supply.
For most plants in natural ecosystems, nitrogen is the key factor limiting
plant growth (Hall et al., 2011). Thus, nitrogen addition usually causes
plants to absorb more N. However, extreme drought could prevent plants from
absorbing N even under high N supply. In the present experiment, N supply
was found to have an effect on N contents in H. ammodendron. Relative to the control
treatment (W0N0), N contents increased with N supply under low N addition
but remained unchanged under high addition (Tables S1, S2). Nitrogen is the main
constituent of Rubisco (ribulose – 1,5 – bisphosphate carboxylase oxygenase)
and chlorophyll in plants. Thus, chlorophyll a was found to have a similar
pattern as N contents under water and N supply. Chlorophyll a was higher in
W0N1 than W0N0, and there was no difference in chlorophyll a between W0N0
and W0N2 (Table S1). Increasing chlorophyll contents in W0N1 should lead to
the increase in photosynthetic rate (A). However, different from our
prediction, one-way ANOVA suggested that A in W0N1 did not differ
from that in W0N0 and that A in W0N2 was lower than that in W0N0 (Fig. 2a). Two-way ANOVA showed that N addition had an influence on A
(Table 1). Both the analyses suggested that N supply played a negative role
in A. These results might be associated with the extremely high light
intensity at the study site. Due to the high light intensity, photosynthetic
rate might not be correlated with chlorophyll contents (Gabrielsen, 1948).
The negative effect of N supply on A led to the decrease in the consumption
of intercellular CO2. Consequently, ci/ca ratio increased
with N supply (Fig. 2d, Table 1). Therefore, the variations in
ci/ca ratio with N addition could not account for the unchanged
pattern in δ13C under N supply (Fig. 1).
The co-application of water and nitrogen had a negative effect on A but no
effect on gs (W0N0 vs. W1N1, W1N2, Fig. 2a, b). The responses of A and
gs to the co-application of water and nitrogen resulted in an increase
in ci/ca ratio (Fig. 2d). Since δ13C remained
unchanged under the co-application of water and nitrogen (Fig. 1),
ci/ca ratio could not also explain the observed δ13C
response to the co-application of water and nitrogen.
In summary, the unchanged δ13C across treatments was not
dependent on the ci/ca ratio in H. ammodendron (Fig. 4e). The observed δ13C stability across treatments might be associated with the φ value and carbonic anhydrase (CA) in H. ammodendron. For C4 plants, the
relationship between carbon isotope discrimination (Δ≈δ13Cair-δ13Cplant; see Eq. 2) and
ci/ca ratio is controlled by φ values (Ellsworth and
Cousins, 2016; Ellsworth et al., 2017; Farquhar, 1983; Wang et al., 2008).
Some studies suggested that φ value was stable for a given species
under a wide range of environmental conditions (Henderson et al., 1992; Wang
et al., 2008; Cernusak et al., 2013). However, other studies had different
conclusions that φ value was influenced by irradiation (Bellasio
and Griffiths, 2014; Kromdijk et al., 2010; Pengelly et al., 2010; Ubierna
et al., 2013), temperature (von Caemmerer et al., 2014), water stress
(Fravolini et al., 2002; Gong et al., 2017; Williams et al., 2001; Yang et
al., 2017) and nitrogen supply (Fravolini et al., 2002; Meinzer and Zhu,
1998; Yang et al., 2017). In the current study, the φ value of H. ammodendron
remained unchanged across six treatments (Fig. 5), and two-way ANOVA suggested that water supply and N supply had no effect on φ (Table 1). Therefore, the φ value of H. ammodendron was insensitive to water
and N addition in this study. Even if the φ value remains stable,
the relationship between Δ and ci/ca ratio is also
associated with the magnitude of the φ value. Cernusak et al. (2013) predicted that when the φ value is greater than 0.37, the
correlation between Δ and ci/ca ratio is positive;
conversely, when the φ value is less than 0.37, the correlation is
negative. In particular, when the φ value is equal to 0.37, there will
be no correlation between them, because the coefficient ([b4+φ(b-s)-a] in Eq. 2) of ci/ca ratio equals 0
(Cernusak et al., 2013). The φ value calculated from the average
δ13Cair ranged from 0.32 to 0.59 with a mean value of 0.45
in the present study. Thus, the correlation between Δ and
ci/ca in H. ammodendron should be positive based on the prediction by Cernusak et
al. (2013). Δ always changes in the opposite direction to δ13Cplant changes according to Eq. (2); thus, a negative
relationship between δ13Cplant and ci/ca is
expected. In fact, this study observed no correlation between δ13C and ci/ca in H. ammodendron (Fig. 4e); this indicates that φ
was not the driver of the observed δ13C pattern in H. ammodendron. However,
the measured δ13C represents the fixed carbon isotope
composition throughout the assimilation branch formation period, which
usually spans at least several weeks. And the measured ci/ca is an
instant indicator. As a result, there were some uncertainties in the
calculation of φ value using Eq. (2) based on the measured δ13C and ci/ca. In addition, the mean φ values
calculated from the minimum and maximum δ13Cair were 0.35
and 0.55, respectively, suggesting that the φ value of H. ammodendron might be
close to 0.37, which led to the observed insensitive response of δ13C to water and N addition.
The enzymatic activity of CA may be another mechanism behind the unchanged
δ13C across treatments. Cousins et al. (2006) suggested that
enzymatic activity of CA affects carbon isotope discrimination in most
C4 plants because CA can result in the parameter b4 changes (see
Eq. 2). But in the traditional view, the parameter b4 was a constant.
However, it is only true when the ratio of PEP carboxylation rate to the
CO2 hydration rate (Vp/Vh) is equal to zero, which is caused
by a high CA activity. If Vp/Vh is not zero, b4 will change
and be controlled by Vp/Vh (Cousins et al., 2006). Previous
studies reported that CA activity was low in most C4 plants (Cousins et
al., 2006; Gillon and Yakir, 2000, 2001; Hatch and Burnell, 1990). Thus, CA
activity in H. ammodendron might also be low, leading to the change in b4 with
Vp/Vh, and thus δ13C. Cousins et al. (2006) added
Vp/Vh into the discrimination pattern of C4 plants and
predicted that at a given φ value, when the Vp/Vh is 0 or
1, the correlation between Δ and ci/ca ratio is negative
or positive, respectively. Since CA activity is low in most C4 plants,
and the Vp/Vh always ranges from 0 to 1, we speculate that no
correlation between Δ and ci/ca ratio may also occur when
the Vp/Vh is a certain value between 0 and 1. The irrelevance of
Δ and ci/ca ratio also means that δ13Cplant is not related to ci/ca ratio due to the
negative correlation between Δ and δ13Cplant
according to Eq. (2). Thus, the uncorrelated pattern between δ13C and ci/ca ratio in H. ammodendron might be related to this specific
Vp/Vh value due to low CA activity.
In addition, the unchanged δ13C across treatments may also be
controlled by the water sources of H. ammodendron. A previous study has found that the root
of H. ammodendron can be inserted into the soil layer deeper than 3 m (Sheng et al.,
2004), which made it easy to uptake groundwater. Therefore, H. ammodendron may be less
sensitive to water addition. However, a study conducted in the same region
has found that the shallow soil water (0–40 cm) and groundwater are two
important water sources for H. ammodendron (Dai et al., 2014), and another study has
reported that water addition resulted in an increase in soil water contents
in the shallow soil layer (Cui, 2018). Moreover, gas exchange changed across
treatments in the present study (Fig. 2). Thus, the utilization of
groundwater by H. ammodendron may be one of the reasons why its δ13C was not
sensitive to water and N addition, but it should not be the main reason.
Whether foliar δ13C of C4 plants can indicate their WUE is
still controversial. Henderson et al. (1992) found that δ13C of
10 C4 species has negative correlation with their WUE. Although this
result was just opposite to a positive relationship between δ13C and WUE for C3 plants (Farquhar, 1983; Duquesnay et al.,
1998; Feng, 1998), it is proof that δ13C of C4 plants
can indicate their WUE. In the work of Henderson et al. (1992), they found
that the φ values in 10 C4 species were around 0.21 over a
range of irradiance and leaf temperature. According to the suggestion by
Cernusak et al. (2013) that Δ is negatively related to
ci/ca ratio when φ value is less than 0.37, the
δ13C of 10 C4 species has a positive correlation with
ci/ca ratio. In general, under fixed ambient CO2
concentration, WUE is always negatively correlated with ci/ca
ratio (see Eqs. 4 and 6). This is why a negative relationship between
δ13C and WUE was observed for the 10 C4 species. The
present study showed that ins-WUE and int-WUE both had no correlation with
δ13C in H. ammodendron (Fig. 4a, c), which was different from the results
published by Henderson et al. (1992). In general, ci/ca ratio is
the link between WUE and δ13C. As mentioned above, if the
φ value equals 0.37 and/or the activity of CA is very low,
δ13C would not correlate to ci/ca ratio and thus
leads to the uncorrelation between δ13C and WUE. In addition,
the different timescales of δ13C, ins-WUE and int-WUE may also
result in this uncorrelation. As mentioned above, the measured δ13C represents the long-term fixed carbon isotope composition (at
least several weeks). And the values of ins-WUE and int-WUE were calculated
from the gas exchange of a short-term measurement, which lasted only a few
minutes. Therefore, this difference in timescale may also drive the
uncorrelation between δ13C and WUE. Although the defects in
measurements could introduce some uncertainty in the observed relationship
between δ13C and WUE, δ13C remained stable under
water and nitrogen addition (Fig. 1, Table 1), while the measured ins-WUE
and int-WUE were higher in the control treatment (W0N0) than other treatments
(Fig. 3), suggesting water and N supply had a significant effect on WUE
(Table 1). These results indirectly confirmed that δ13C of H. ammodendron
could not indicate its WUE.
The present study has found that δ13C of H. ammodendron could not be used as
an indicator of its WUE. Although this conclusion cannot be analogous to all
C4 plants, the present study has important implications for the
understanding of physiological responses of desert plants to future changes
in precipitation and atmospheric N deposition. H. ammodendron is a dominant species in
Asian desert, which has a great effect on the stabilization of sand dunes,
the survival and development of understory plants, and the structure and
function of desert ecosystems (Sheng et al., 2005; Su et al., 2007; Cui et
al., 2017). Thus, H. ammodendron is widely distributed in desert areas, and the prediction
of its drought adaptation is crucial in desert ecosystems.
Conclusion
Global changes including precipitation and atmospheric N deposition have
been proven to have an important influence on ecosystems, especially for
arid ecosystems. The present study showed that water and N addition had
little effect on the δ13C values and the degree of
bundle-sheath leakiness (φ) of H. ammodendron but played an important role in
the change of its gas exchange and water use efficiency (WUE). In addition,
different patterns of instantaneous WUE (ins-WUE), intrinsic WUE (int-WUE)
and δ13C across treatment and no correlation between
instantaneous WUE (ins-WUE) and δ13C and between intrinsic WUE
(int-WUE) and δ13C have been found in this study, suggesting
that δ13C of H. ammodendron could not indicate its WUE. This result was
caused by the lack of correlation between δ13C and the
ratio of intercellular to ambient CO2 concentration (ci/ca),
which might be associated with the degree of bundle-sheath leakiness
(φ) or the low activity of carbonic anhydrase (CA). Thus, the
current experiment implies that the availability of δ13C as the
indicator of WUE could be not universal for C4 species.
Data availability
The datasets analyzed in this paper are not publicly available.
Requests to access the datasets should be directed to
gawang@cau.edu.cn.
The supplement related to this article is available online at: https://doi.org/10.5194/bg-18-2859-2021-supplement.
Author contributions
GW and JL designed the experiment and modified the manuscript. ZC
designed and executed the experiment and wrote the manuscript. XL
designed the experiment. XC executed the experiment. YH executed the
experiment.
Competing interests
The authors declare that they have no conflict of interest.
Acknowledgements
This research was supported by the Chinese National Basic Research Program
(no. 2014CB954202) and a grant from the National Natural Science Foundation
of China (no. 41772171). We are grateful for the support from the Fukang
Observation Station of Desert Ecology and Xinjiang Institute of Ecology and
Geography, Chinese Academy of Sciences. We would like to thank Ma Yan for analyzing
stable carbon isotope ratios in the Isotope Lab at the College of Resources
and Environment, China Agricultural University.
Financial support
This research was supported by the Chinese National Basic Research Program (no. 2014CB954202) and a grant from the National Natural Science Foundation of China (no. 41772171).
Review statement
This paper was edited by Aninda Mazumdar and reviewed by three anonymous referees.
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