Evaluating the response of δ¹³C in Haloxylon ammodendron, a dominant C₄ species in Asian desert ecosystems, to water and nitrogen addition as well as the availability of its δ¹³C as an indicator of water use efficiency

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 (c_i $/$ c_a), 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.

1 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 ($c_{\mathrm{i}}/c_{\mathrm{a}}$), which reflects the balance between inward CO₂ diffusion rate, regulated by stomatal conductance (g_s), 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 c_i $/$ c_a 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 (HCO${}_{\mathrm{3}}^{-}$). 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 c_i $/$ c_a ratio and δ¹³C and between c_i $/$ c_a ratio and WUE (Ehleringer and Cerling, 1995). For C₃ plants, δ¹³C always decreases with an increase in c_i $/$ c_a ratio; but for C₄ plants, the correlation between δ¹³C and c_i $/$ c_a 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 c_i $/$ c_a 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.

2 Materials and methods

2.1 Definitions and basic equations

Stable carbon isotopic ratio (δ¹³C) of natural materials is expressed as

$$\begin{array}{}\text{(1)}& {\mathit{\delta }}^{\mathrm{13}}\mathrm{C}\left(\mathrm{‰}\right)=\left[{\displaystyle \frac{{(}^{\mathrm{13}}\mathrm{C}{/}^{\mathrm{12}}\mathrm{C}{)}_{\mathrm{sample}}}{{(}^{\mathrm{13}}\mathrm{C}{/}^{\mathrm{12}}\mathrm{C}{)}_{\mathrm{standard}}}}-\mathrm{1}\right]\times \mathrm{1000},\end{array}$$

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:

$$\begin{array}{}\text{(2)}& \begin{array}{rl}\mathrm{\Delta }& ={\displaystyle \frac{{\mathit{\delta }}^{\mathrm{13}}{\mathrm{C}}_{\mathrm{air}}-{\mathit{\delta }}^{\mathrm{13}}{\mathrm{C}}_{\mathrm{plant}}}{\mathrm{1}+{\mathit{\delta }}^{\mathrm{13}}{\mathrm{C}}_{\mathrm{plant}}/\mathrm{1000}}}\approx {\mathit{\delta }}^{\mathrm{13}}{\mathrm{C}}_{\mathrm{air}}-{\mathit{\delta }}^{\mathrm{13}}{\mathrm{C}}_{\mathrm{plant}}\\ & =a+\left[b_{\mathrm{4}}+\mathit{\phi }\left(b-s\right)-a\right]{\displaystyle \frac{c_{\mathrm{i}}}{c_{\mathrm{a}}}},\end{array}\end{array}$$

where δ¹³C_plant and δ¹³C_air 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 HCO${}_{\mathrm{3}}^{-}$ in mesophyll cells, and HCO${}_{\mathrm{3}}^{-}$ 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 c_i $/$ c_a is the ratio of intercellular to ambient CO₂ concentration. Eq. (2) can be transformed into the following format:

$$\begin{array}{}\text{(3)}& {\mathit{\delta }}^{\mathrm{13}}{\mathrm{C}}_{\mathrm{plant}}=-\left[b_{\mathrm{4}}+\mathit{\phi }\left(b-s\right)-a\right]{\displaystyle \frac{c_{\mathrm{i}}}{c_{\mathrm{a}}}}+{\mathit{\delta }}^{\mathrm{13}}{\mathrm{C}}_{\mathrm{air}}-a.\end{array}$$

According to Eq. (3), if the coefficient [b₄+φ (b−s) − a] is greater than 0, δ¹³C decreases with increasing c_i $/$ c_a; if this coefficient is lower than 0, δ¹³C increases with increasing c_i $/$ c_a.

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

$$\begin{array}{}\text{(4)}& \text{ins-WUE}=A/E=(c_{\mathrm{a}}-c_{\mathrm{i}})/\mathrm{1.6}v=c_{\mathrm{a}}(\mathrm{1}-c_{\mathrm{i}}/c_{\mathrm{a}})/\mathrm{1.6}v,\end{array}$$

where A is photosynthetic rate, E is transpiration rate and v is calculated by

$$\begin{array}{}\text{(5)}& v=(e_{\mathrm{i}}-e_{\mathrm{a}})/p,\end{array}$$

where e_i and e_a are the water vapor pressure inside and outside the leaves, and p is the atmospheric pressure.

The definition of int-WUE is

$$\begin{array}{}\text{(6)}& \text{int-WUE}=A/g_{\mathrm{s}}=(c_{\mathrm{a}}-c_{\mathrm{i}})/\mathrm{1.6}=c_{\mathrm{a}}(\mathrm{1}-c_{\mathrm{i}}/c_{\mathrm{a}})/\mathrm{1.6},\end{array}$$

where g_s is stomatal conductance.

2.2 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.

2.3 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.

2.4 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 (g_s), transpiration rate (E), the ambient CO₂ concentration (c_a) and the intercellular CO₂ concentration (c_i). 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.

2.5 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.

2.6 Measurements of plant δ¹³C, plant N and chlorophyll contents

The δ¹³C and N measurements were performed on a Delta^Plus 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:

$$\begin{array}{}\text{(7)}& {\displaystyle }\mathrm{Chlorophyll}a(\mathrm{mg}/\mathrm{L})=\mathrm{13.95}\times \mathrm{OD}\mathrm{665}-\mathrm{6.88}\times \mathrm{OD}\mathrm{649},\text{(8)}& {\displaystyle }\mathrm{Chlorophyll}b(\mathrm{mg}/\mathrm{L})=\mathrm{24.96}\times \mathrm{OD}\mathrm{649}-\mathrm{7.32}\times \mathrm{OD}\mathrm{665},\end{array}$$

where OD665 and OD649 are the absorbency under the wave lengths of 665 and 649 mm, respectively.

2.7 Calculation of the degree of bundle-sheath leakiness

The degree of bundle-sheath leakiness (φ) was calculated by the transformation of Eq. (2):

$$\begin{array}{}\text{(9)}& \begin{array}{rl}\mathit{\phi }& =\left({\displaystyle \frac{({\mathit{\delta }}^{\mathrm{13}}{\mathrm{C}}_{\mathrm{air}}-{\mathit{\delta }}^{\mathrm{13}}{\mathrm{C}}_{\mathrm{plant}})/(\mathrm{1}+{\mathit{\delta }}^{\mathrm{13}}{\mathrm{C}}_{\mathrm{plant}}/\mathrm{1000})-a}{c_{\mathrm{i}}/c_{\mathrm{a}}}}+a-b_{\mathrm{4}}\right)\\ & /\left(b-s\right).\end{array}\end{array}$$

In this equation, parameters a, b₄, b and s are constant, while δ¹³C_plant and c_i $/$ c_a are the measured values of our samples. We did not measure the δ¹³C_air at our study site, so we had to use an approximation of the δ¹³C_air 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 δ¹³C_air 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 δ¹³C_air has large diurnal and seasonal variations, we used the published range of δ¹³C_air 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 δ¹³C_air of the time period, which is the main growing season for H. ammodendron. Finally, the δ¹³C_air used in the calculation ranged from −10.52 ‰ to −9.01 ‰ with an average of −9.77 ‰.

2.8 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 c_i $/$ c_a in H. ammodendron.

3 Results

3.1 Plant 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).

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. φ_min, φ_ave and φ_max represent the φ values calculated from the minimum,
average and maximum δ¹³C_air .^*, ${}^{**}$ and ${}^{***}$ indicate a significant influence. W ⋅ N represents the interaction between water addition and N addition.

Figure 1 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).

3.2 Gas exchange and WUE under water and nitrogen addition

Photosynthetic rate (A), stomatal conductance (g_s), transpiration rate (E) and c_i $/$ c_a 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, g_s, E and c_i $/$ c_a, Fig. 2). Two-way ANOVA suggested that water addition had exerted an effect on c_i $/$ c_a (p < 0.01), that N additions influenced A (p < 0.01) and c_i $/$ c_a (p= 0.009), and that the interaction between water and N supply played a role in g_s (p < 0.001), E (p < 0.001) and c_i $/$ c_a (p < 0.001, Table 1).

Figure 2 Variations in photosynthetic rate (a), stomatal conductance (b), water use efficiency (c) and c_i $/$ c_a (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).

Figure 3 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).

3.3 Correlations among δ¹³C, WUE and c_i $/$ c_a ratio

In order to test whether δ¹³C in H. ammodendron can indicate WUE, the relationships among δ¹³C, ins-WUE, int-WUE and c_i $/$ c_a 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 c_i $/$ c_a ratio (p= 0.18, Fig. 4e). However, there was a negative correlation between ins-WUE and c_i $/$ c_a ratio (p < 0.001, Fig. 4b) and between int-WUE and c_i $/$ c_a ratio (p < 0.001, Fig. 4d).

Figure 4 Correlations of ins-WUE vs. δ¹³C (a), ins-WUE vs. c_i $/$ c_a (b), int-WUE vs. δ¹³C (c), int-WUE vs. c_i $/$ c_a (d) and δ¹³C vs. c_i $/$ c_a (e) of assimilating branches of Haloxylon ammodendron.

3.4 The degree of bundle-sheath leakiness under water and nitrogen addition

The φ value calculated from the minimum δ¹³C_air ranged from 0.16 to 0.50 with a mean value of 0.35; the φ value calculated from the maximum δ¹³C_air ranged from 0.44 to 0.70 with a mean value of 0.55, and the φ value calculated from the average δ¹³C_air 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 δ¹³C_air across treatments (p= 0.60 for the φ calculated from the minimum δ¹³C_air, p= 0.77 for the φ calculated from the average δ¹³C_air and p= 0.90 for the φ calculated from the maximum δ¹³C_air, Fig. 5). Two-way ANOVA suggested that φ was not affected by water addition (p= 0.46 for the φ calculated from the minimum δ¹³C_air, p= 0.64 for the φ calculated from the average δ¹³C_air and p= 0.98 for the φ calculated from the maximum δ¹³C_air), N addition (p= 0.65 for the φ calculated from the minimum δ¹³C_air, p=  0.60 for the φ calculated from the average δ¹³C_air and p= 0.55 for the φ calculated from the maximum δ¹³C_air) or their interaction (p= 0.30 for the φ calculated from the minimum δ¹³C_air, p= 0.52 for the φ calculated from the average δ¹³C_air and p = 0.87 for the φ calculated from the maximum δ¹³C_air, Table 1).

Figure 5 Variations in φ calculated by Eq. (9) using the minimum (Min), average (Ave) and maximum (Max) values of δ¹³C_air across water (W) and nitrogen (N) additions. The box represents the mean value of four replicates with error bars denoting the standard error (SE).

4 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 c_i $/$ c_a ratio, which reflects the balance between stomatal conductance (g_s) and photosynthetic rate (A) (Farquhar and Richards, 1984). Stomatal conductance (g_s) usually increases with increasing water availability under water addition. Although two-way ANOVA suggested that water addition had no effect on both A and g_s (Table 1), one-way ANOVA showed that g_s was higher in W1N0 than that in W0N0 (Fig. 2b), indicating that water addition had a positive effect on g_s under ambient N conditions. Increasing g_s under water supply will lead to the rise of intercellular CO₂ because of the decrease in diffusional resistance to CO₂. As a result, c_i $/$ c_a ratio was observed to increase with increasing moisture (Fig. 2d, Table 1). However, δ¹³C remained stable under water addition (Fig. 1, Table 1). Thus, c_i $/$ c_a 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, c_i $/$ c_a ratio increased with N supply (Fig. 2d, Table 1). Therefore, the variations in c_i $/$ c_a 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 g_s (W0N0 vs. W1N1, W1N2, Fig. 2a, b). The responses of A and g_s to the co-application of water and nitrogen resulted in an increase in c_i $/$ c_a ratio (Fig. 2d). Since δ¹³C remained unchanged under the co-application of water and nitrogen (Fig. 1), c_i $/$ c_a 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 c_i $/$ c_a 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≈ δ¹³C_air−δ¹³C_plant; see Eq. 2) and c_i $/$ c_a 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 c_i $/$ c_a 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 c_i $/$ c_a 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 ([$b_{\mathrm{4}}+\mathit{\phi }(b-s)-a$] in Eq. 2) of c_i $/$ c_a ratio equals 0 (Cernusak et al., 2013). The φ value calculated from the average δ¹³C_air ranged from 0.32 to 0.59 with a mean value of 0.45 in the present study. Thus, the correlation between Δ and c_i $/$ c_a in H. ammodendron should be positive based on the prediction by Cernusak et al. (2013). Δ always changes in the opposite direction to δ¹³C_plant changes according to Eq. (2); thus, a negative relationship between δ¹³C_plant and c_i $/$ c_a is expected. In fact, this study observed no correlation between δ¹³C and c_i $/$ c_a 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 c_i $/$ c_a 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 c_i $/$ c_a. In addition, the mean φ values calculated from the minimum and maximum δ¹³C_air 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 ($V_{\mathrm{p}}/V_{\mathrm{h}}$) is equal to zero, which is caused by a high CA activity. If $V_{\mathrm{p}}/V_{\mathrm{h}}$ is not zero, b₄ will change and be controlled by $V_{\mathrm{p}}/V_{\mathrm{h}}$ (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 $V_{\mathrm{p}}/V_{\mathrm{h}}$, and thus δ¹³C. Cousins et al. (2006) added $V_{\mathrm{p}}/V_{\mathrm{h}}$ into the discrimination pattern of C₄ plants and predicted that at a given φ value, when the $V_{\mathrm{p}}/V_{\mathrm{h}}$ is 0 or 1, the correlation between Δ and c_i $/$ c_a ratio is negative or positive, respectively. Since CA activity is low in most C₄ plants, and the $V_{\mathrm{p}}/V_{\mathrm{h}}$ always ranges from 0 to 1, we speculate that no correlation between Δ and c_i $/$ c_a ratio may also occur when the $V_{\mathrm{p}}/V_{\mathrm{h}}$ is a certain value between 0 and 1. The irrelevance of Δ and c_i $/$ c_a ratio also means that δ¹³C_plant is not related to c_i $/$ c_a ratio due to the negative correlation between Δ and δ¹³C_plant according to Eq. (2). Thus, the uncorrelated pattern between δ¹³C and c_i $/$ c_a ratio in H. ammodendron might be related to this specific $V_{\mathrm{p}}/V_{\mathrm{h}}$ 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 c_i $/$ c_a ratio when φ value is less than 0.37, the δ¹³C of 10 C₄ species has a positive correlation with c_i $/$ c_a ratio. In general, under fixed ambient CO₂ concentration, WUE is always negatively correlated with c_i $/$ c_a 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, c_i $/$ c_a 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 c_i $/$ c_a 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.

5 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 (c_i $/$ c_a), 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.