Changes in precipitation and atmospheric N deposition affect the correlation between N, P and K but not the coupling of water-element in Haloxylon ammodendron.

Global changes in precipitation and atmospheric N deposition affect the geochemical cycle of the element and its hydrological cycle in the ecosystem. It may also affect the relationship between plant water use efficiency (WUE) and nutrients, as well as the relationship between plant nutrients. Desert ecosystems are vulnerable to global changes. Haloxylon ammodendron is the dominant species in the Asian desert. Revealing the variations in these relationships in H. ammodendron with precipitation and N deposition will enhance our understanding of the responses of plants to global change in terms of trade-off strategies of nutrient absorption, water and element geochemical cycles in desert ecosystems. Thus, we conducted field experiments with different amounts of water and N. This study showed that WUE of H. ammodendron was not correlated with nitrogen content (N), phosphorus content (P), and potassium content (K) when water and N supply were varied (p > 0.05 for WUE vs. N, P, and K), suggesting lack of coupling between water use and nutrient economics. This result was associated with the lack of correlation between plant nutrients and gas exchang in H. ammodendron. However, water addition, N addition and the interaction between both of them all played a role in the correlation between plant N, P and K owing to their different responses to water and N supplies. This indicates that global changes in precipitation and N deposition will affect N, P and K geochemical cycles in the Asian deserts dominated by H. ammodendron, and drive changes in the relationships between plant nutrients, resulting in changes in the trade-off strategy of plant absorption of N, P, and K.

Introduction

Atmospheric N deposition has continued to rise since the Industrial Revolution [1-3], and the pattern of global rainfall has also changed [4, 5]. These changes significantly affected plant resource utilization, hydrological cycles, and geochemical cycles of elements. Plant water economy is closely associated with water geochemical cycle, and nutrition economy is closely related to element geochemical cycle. To resist environmental changes, the adjustment of nitrogen, phosphorus, and potassium economies and water economy is an important strategy for resource utilization in plants. Nitrogen content (N), phosphorus content (P), and potassium content (K) in plants can serve as surrogates for the nitrogen, phosphorus, and potassium economies [6]. Many studies have demonstrated that plants N, P, and K are closely related with each other [7-11]. Water use efficiency (WUE) is the main indicator of plant water economy, and it is associated with gas exchange including photosynthetic rate (A), transpiration rate (E) and stomatal conductance (g). Plant N is positively correlated with A because it is the main constituent of Rubisco (ribulose-1,5-bisphosphate carboxylase oxygenase) and chlorophyll in plants [12-14]. Phosphorus is required for many compounds involved in photosynthesis [15]; therefore, plant P is positively correlated with some parameters related to photosynthesis, such as maximal Rubisco carboxylation rate and maximum electron transport rate [16-18]. Potassium plays a crucial role in adjusting stomata, osmotic pressure and enzyme activity [19-21], and thus strongly controls the changes in gs and E. Therefore, WUE is expected to be related to plant N, P and K, and water use should be linked to plant N, P, and K economy [6]. Several studies have reported tight coupling between WUE and plant N, P and K [6, 22–27]. However, other investigations did not observe these couplings [23, 26, 28, 29]. The relationship between plant WUE and nutrients and the relationship between plant N, P and K reflect the coupling between water cycle and the geochemical cycles of these elements in ecosystems along with the resource-use strategies adopted by plants [30, 31].

Changes in rainfall and N deposition may also affect the relationship between plant water use and nutrient use, and the relationship between different nutrient elements. Two meta-analyses have found that global change, including change in N deposition and precipitation, have resulted in variations in plant N/P ratio and N/K ratio [32, 33], indicating that plant element coupling has been affected by changes in rainfall and N deposition. Under increasing precipitation, plants will take up more nutrients [22], whereas WUE will decrease [34]. Increasing N deposition promotes photosynthesis [12-14], causing plant WUE to increase. Despite this, due to the dilution effect of biomass, plant N, P, and K may not increase. Therefore, with N deposition and precipitation changes, the direction of changes in WUE and plant N, P, and K may be different, which will lead to changes in the correlation between WUE and plant N, P, and K. However, at present, only a few studies have investigated the influence of N deposition and precipitation changes on the relationship between WUE and plant N, P, and K.

Due to extreme drought and barrenness, desert ecosystems are vulnerable to environmental changes [35, 36]. Rainfall changes and elevated atmospheric N deposition are two important factors influencing the availabilities of water and N in deserts [37]. Haloxylon ammodendron is a dominant species in desert regions, particularly in Asian deserts. It plays an important role in the stabilization of sand dunes, the survival and development of understory plants, and the structure and function of desert ecosystems [38-40]. Given the universal plant WUE-nutrient coupling and the mutual relationships between plant N, P, and K [6.7.27], and the possible impact of changes in precipitation and N deposition on these couplings [32, 33], we hypothesized that: (1) plant WUE-nutrient coupling and couplings among plant N, P, and K should occur in H. ammodendron; (2) the correlation between plant nutrients should vary with changes in rainfall and atmospheric N deposition for H. ammodendron; and (3) changes in rainfall and atmospheric N deposition also have an effect on these correlations between WUE and N, P, and K for H. ammodendron. To test our hypotheses, we conducted a field experiment by varying the supply of water and nitrogen in the southern Gurbantunggut Desert in Xinjiang Uygur Autonomous Region, China. We measured WUE, N, P and K of the assimilating branches of H. ammodendron. We hope this study can enhance the understanding of the resource-use strategies adopted by plants growing in desert ecosystems and the responses of water and element geochemical cycles to changes in N deposition and precipitation in desert ecosystems.

Materials and methods

Study site

This study was conducted at the Fukang Station of Desert Ecology, Chinese Academy of Sciences, on the southern edge of the Gurbantunggut Desert (44°26′ N, 87°54′ E) in northwestern China. The altitude of our study site is 436.8 m above average sea level (a.s.l.). It has a typical continental arid, temperate climate with a hot summer and cold winter. The mean annual temperature is 7.1 ºC, and the mean annual precipitation is 215.6 mm, with potential evaporation of about 2000 mm. The soil type is gray desert soil (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) ranged from 1.63% to 1.76%, 13.79% to 14.15%, 55.91% to 56.21% and 20.65% to 23.23%, respectively [41]. The soil is highly alkaline (pH = 9.55 ± 0.14) with low fertility. The vegetation is dominated by Haloxylon ammodendron and Haloxylon persicum with approximately 30% coverage. Herbs include ephemerals, annuals, and small perennials, covering approximately 40% [42]. Although the coverage of the two Haloxylon species is slightly lower than that of herbs, the biomass of the former is much larger than that of the latter. This is because Haloxylon plants are small trees with an average height of 1.5 m, whereas the latter are very low herbaceous plants. Biological soil crusts are distributed widely in the soil between herbs and Haloxylon, with approximately 40% coverage [43].

Experimental design

A field experiment with a completely randomized factorial combination of water and nitrogen has been conducted since 2014. Two water addition levels (0, 60 mm·yr−1; W0, W1) were chosen based on the fact that precipitation was predicted to increase by 30% in northern China in the next 30 years [44]. Three levels of N addition (0, 30, 60 kg N·ha−1·yr−1; N0, N1 and N2) were chosen based on the fact that N deposition has reached 35.4 kg N·ha−1·yr−1 in a nearby city, Urumqi [40] and is expected to double by 2050 relative to the early 1990s [45]. Therefore, six treatments (W0N0, W0N1, W0N2, W1N0, W1N1, and W1N2) were used in this experiment. Four replicates of each treatment were set, resulting in a total of 24 plots. The plots were distributed in a lowland area between two dunes. Each plot was 1.5 m×1.5 m with a H. ammodendron enclosed in the center. The average height and coverage of an individual H. ammodendron were 1.5 m and 1.9 m2, respectively; they did not vary significantly across the plots. To simulate natural rainfall and N deposition, water and N additions were applied in equal amounts, twelve times; once every week in April, July and September, with 5 mm·m-2 of water and 2.5, or 5 kg N·ha−1 [40]. Water was added with a sprinkler kettle, irrigating over the canopy of H. ammodendron. The added N was NH4NO3 because the NH4+-N content was basically equal to the NO3-N content in the local atmospheric N deposition. To improve absorption of plants and better simulate atmospheric N deposition, we dissolved NH4NO3 in 0.5 L·m-2 water and sprayed it to the plot uniformly with a sprayer. The control treatment (N0) was sprayed with the same amount of water. The time and frequency of adding N and water remained the same.

Sampling of H. ammodendron and soil

Sampling was conducted in July of 2017. We collected the assimilating branches of H. ammodendron as our samples. Eight pieces of assimilating branches were obtained from each individual, with two pieces of assimilating branches being taken from each of the four cardinal directions with respect to the derection of full irradiance. All assimilating branches from the same plot were combined into a single sample. The topsoil (0–5 cm) of each treatment plot was also sampled. After sampling, the fresh soil samples were sieved through 2mm to remove the rocks and then air-dried in the laboratory.

Measurements of N, P and K of H. ammodendron

The N content (N) of the assimilating branch samples was measured on a DeltaPlus XP mass spectrometer (Thermo Scientific, Bremen, Germany) coupled with an automated elemental analyzer (Flash EA1112, CE Instruments, Wigan, UK) in the continuous flow mode. The standard deviation for the N measurements was 0.1%. The P and K content (P and K) of the assimilating branch samples was measured using an inductively coupled plasma source mass spectrometer (ICP-MS) after HNO3-H2O2 digestion.

Measurements of soil N, P contents and Olsen-P

Soil total N (STN) of each soil sample was measured using an automated elemental analyzer (VARIO EL cube). The standard deviation for the measurements was 0.05%. The soil total P (STP) of each soil sample was measured on a spectrophotometer by Mo-Sb colorimetry after HClO-H2SO4 digestion. In addition, we measured the soil Olsen-P (SOP) on a spectrophotometer by Mo-Sb colorimetry after NaHCO3 extraction.

Statistical analysis

Statistical analyses were conducted using the SPSS software (SPSS for Windows, Version 20.0, Chicago, IL, USA). One-way analysis of variance (ANOVA) and two-way ANOVA were used to compare the differences in plant N, P and K and soil N and P between each treatment. The least significant difference (LSD) test was conducted after each one-way ANOVA analysis to determine the difference in each index between every two treatments. Linear-regression analysis and Pearson analysis were used to determine the correlation between plants N, P, and K and between water use efficiency (WUE), including instantaneous WUE (ins-WUE) and intrinsic WUE (int-WUE), and plants N, P, K in H. ammodendron in all samples. Ins-WUE was calculated by the quotient of A and E, while int-WUE was calculated by the quotient of A and g. The data of WUE and gas exchange came from our published paper [46].

Results

Changes in plant N, P, and K and soil N, P across treatments

Plant N, P, and K concentrations in the assimilating branches ranged from 17.40 mg g-1 to 30.68 mg g-1, 0.73 mg g-1 to 1.59 mg g-1 and 13.98 mg g-1 to 39.76 mg g-1, respectively. One-way ANOVA analyses showed that plant N in W0N1 was significantly higher than that in other treatments (all p < 0.05 by LSD test, Fig 1a). Plant P was significantly higher in W0N1 than in W0N2 (p = 0.046), W1N0 (p = 0.035) and W1N1 (p = 0.017 by LSD test, Fig 1b). Plant K was significantly higher in W0N1 than in W0N2 (p = 0.006 by LSD test), W1N0 (p = 0.025 by LSD test), W1N1 (p = 0.001 by LSD test) and W1N2 (p = 0.043 by LSD test, Fig 1c). Two-way ANOVA analyses suggested that N addition played a role in plant N (Table 1). Water addition affected plant K (Table 1). The interaction between water and N addition had an effect on plant N and K (Table 1).

Fig 1

Variations in plant N content (a), P content (b), K content (c), soil total N content (d), soil total P content (e) and soil Olsen-P content (f) across water (W) and nitrogen (N) additions.

The box represents the mean value of four replicates with error bars denoting the standard error (SE).

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.

	W	N	W*N
N	0.181	0.010*	0.043*
P	0.089	0.819	0.056
K	0.025*	0.508	0.008**
STN	0.298	0.180	0.153
STP	0.619	0.913	0.917
SOP	0.418	0.455	0.644

Note.

*, **indicates a significant influence.

STN, STP and SOP ranged from 0.23 mg g-1 to 1.00 mg g-1, 0.12 mg g-1 to 1.29 mg g-1 and 9.33 mg kg-1 to 37.87 mg kg-1, respectively. One-way ANOVA analyses showed that STN was significantly lower in W0N0 than in W0N2 (p = 0.028 by LSD test, Fig 1d). The STP did not change significantly across treatments (Fig 1e). However, SOP was significantly lower in W0N0 than in W0N2 (p = 0.033 by LSD test, Fig 1f). Two-way ANOVA analyses suggested that water addition, N addition, and their interactions had no significant effect on these three indexes (Table 1).

Variations in the correlation between WUE and elements and the correlation between elements across water additions

For W0 treatments (including W0N0, W0N1 and W0N2), neither ins-WUE nor int-WUE were related to element content (S1 Table in S1 File). There were positive relationships between N and P (Fig 2a, Table 2) and between N and K (Fig 2b, Table 2). However, no correlation between P and K was observed (Fig 2c). For W1 treatments (including W1N0, W1N1 and W1N2), there was no correlation between WUE and element content (all p > 0.05, S1 Table in S1 File). Plant N was not related to P and K (Fig 2a and 2b), whereas K was positively correlated with P (Fig 2c, Table 2).

Fig 2

Correlations of N vs. P (a), N vs. K (b), K vs. P (c) in W0 treatments (include W0N0, W0N1 and W0N2) and W1 treatments (include W1N0, W1N1 and W1N2).

Table 2

The slope of Linear-regression analysis among N, P and K.

Treatment	N vs. P	N vs. K	K vs. P
W0	0.040	0.811	-
W1	-	-	0.024
N0	-	-	-
N1	0.046	1.533	0.022
N2	0.041	-	0.027
W0 and N	0.043	1.257	-
W1 and N	-	-	0.027

Note: W0 treatment includes W0N0, W0N1 and W0N2 treatments, while W1 treatment included W1N0, W1N1 and W1N2 treatments. N0 treatment included W0N0 and W1N0 treatments, N1 treatment included W0N1 and W1N1treatments, while N2 treatment included W0N2 and W1N2 treatments. The W0 and N treatments included W0N1 and W0N2 treatments, whereas W1 and N treatments contained W1N1 and W1N2 treatments. Only the slopes that pass the significance test are shown in Table.

Variations in the correlation between WUE and elements and the correlation between elements across N additions

For N0 treatments (including W0N0 and W1N0), there was no correlation between WUE and the elements, as well as no correlation between plant N, P and K (S1 Table in S1 File, Fig 3a–3c). For N1 treatments (including W0N1 and W1N1) also, WUE was not correlated with nutrients (S1 Table in S1 File), whereas N, P, and K were related to each other (Fig 3a–3c, Table 2). For N2 treatments (including W0N2 and W1N2) also, WUE was not correlated with element content (S1 Table in S1 File). Plant N was positively related to P (Fig 3a, Table 1), but not K (Fig 3b). Plant P was positively correlated with K (Fig 3c, Table 2).

Fig 3

Correlations of N vs. P (a), N vs. K (b), K vs. P (c) in N0 treatments (includes W0N0 and W1N0), N1 treatments (includeW0N1, and W1N1) and N2 treatments (include W0N2 and W1N2).

Variations in the WUE-element correlation and the N-P-K correlations across the interaction of water and N additions

Under the condition of adding N and no adding water (W0 + N treatments, including W0N1 and W0N2), WUE, including ins-WUE and int-WUE, was not related to element content (all p > 0.05, S1 Table in S1 File). N was positively correlated with P and K (Fig 4a and 4b, Table 2), and K had no coupling with P (Fig 4c). Under the treatments of adding both N and water (W1 + N treatments, including W1N1 and W1N2), WUE, including ins-WUE and int-WUE, had no correlation with nutrients (S1 Table in S1 File). N was not related to P and K (Fig 4a and 4b), while K was positively correlated with P (Fig 4c, Table 2).

Fig 4

Correlations of N vs. P (a), N vs. K (b), K vs. P (c) in W0 + N treatments (include W0N1 and W0N2) and N vs. P (d), N vs. K (e), K vs. P (f) in W1 + N treatments (include W1N1 and W1N2).

Discussion

The effect of changes in precipitation and N deposition on the correlation between WUE and elements in H. ammodendron

In the treatments with N or water alone, and in adding both N and water simultaneously, there was no significant correlation between WUE and N, P and K (S1 Table in S1 File). Thus, the result contradicted our hypothesis that there is a coupling between WUE and N, P and K in H. ammodendron. Although previous studies have observed the coupling between WUE and nutrients [6, 22–27], this study showed that this coupling is not a universal pattern. In addition, previous studies have suggested that the correlation between plant water economy and nutrition economy can be used to predict the physiological indicators of some plant that are difficult to monitor in real-time [47-49]. This prediction should not be suitable for H. ammodendron.

Gas exchange is the link between WUE and plants N, P, and K. Since ins-WUE is equal to A divided E, ins-WUE should be positively related to A, and negatively related to E. In addition, given that int-WUE is calculated by the quotient of A and g, int-WUE should have a positive relationship with A and a negative correlation with gs. As previously mentioned, plant N and P are associated with photosynthetic rate (A) [12–14, 16–18]; plant K strongly controls gs and E [50]. In addition, E and gs can regulate plant N, P, and K by affecting the absorption and transportation of these elements by plants. Therefore, plants N, P, and K have been observed to be related to gas exchange and WUE [6, 13, 16, 50]. However, there was no correlation between N, P, K, and gas exchange and no correlation between N, P, K, and WUE in H. ammodendron (S1, S2 Tables in S1 File). The lack of correlation between N, P, K and gas exchange may be related to the resource utilization strategy of H. ammodendron. The leaf economics spectrum (LES) defines a continuum spanning from fast-growing species with a rapid return of the investments in nutrients and carbon in leaves, to slow-growing species with a slower return [7]. Slow-growing species are characterized by a long life span, but low leaf nutrient concentrations, photosynthesis, respiration, and growth rates [47, 51–53]. H. ammodendron is a slow-growing species. It may be because of the slow return on the investment in nutrients that plant N, P, and K were not related to gas exchange. However, this hypothesis needs to be confirmed. In addition, the water source of H. ammodendron could also cause a lack of correlation between N, P, K and gas exchange. The roots of H. ammodendron are exist at depth greater than 3 m into the soil to uptake groundwater [54]; therefore, groundwater is an important source of water for the plant species. The N, P, and K content in the groundwater may be relatively low. Therefore, even if gas exchange changes, the absorption of these elements by H. ammodendron does not necessarily change accordingly. This causes gas exchange to be independent of N, P, and K. Thus, there is no correlation between WUE and N, P, K.

Effects of changes in precipitation and N deposition on the coupling between N, P and K in H. ammodendron

The correlation between plant N, P and K varied with water addition level (Fig 2), N addition level (Fig 3), and their interaction (Fig 4). These results confirmed our hypothesis that there is a relationship between N, P, and K for H. ammodendron and that the relationship is dependent on precipitation and N deposition.

An increase in rainfall promotes plant growth, leading to an increase in the demand for nutrients. However, in that case, plants may require more N and P than K because K is an important element for regulating osmotic pressure in plants. The increase in plant K reduces the water potential in plants, resulting in a decrease in water loss by transpiration. As a result, plants need to take up more K to resist drought stress [55-57]. Therefore, plant demand for K will decrease as precipitation increases. In contrast, the demand for P by H. ammodendron in the present study may be greater than the demand for N under increased precipitation. Ecologists use plant N/P ratio as an indicator of the relative limitation of N versus P in ecosystems, that is, N/P ratios less than 14 generally indicate N limitation, while N/P ratios greater than 16 suggest P limitation [58]. The N/P ratio in H. ammodendron was mostly higher than 16 (S3 Table in S1 File), suggesting that H. ammodendron suffered from P limitation. Thus, H. ammodendron will absorb more P relative to N and K as plant growth is enhanced by increasing precipitation. As a result, changes in precipitation disrupt the relationships between plant N, P and K.

Increased N deposition produces more N to the soil, thus promoting plant growth. As plant growth is promoted, it needs to absorb more N, P, and K. Due to the increase in soil N, plants absorb more N than P and K. As a result, increased N deposition disrupts the relationship between plants N, P, and K. Because both N deposition and precipitation affect the correlation between plant nutrients, it will inevitably lead to the influence of the interaction of N deposition and precipitation on this correlation.

Plants absorb N, P, and K in a specific proportion due to the proportionate requirement of N, P, and K to maintain optimal growth and development. The proportionate absorption of N, P, and K is a trade-off strategy, leading to the relationship between plant N, P, and K. However, this trade-off strategy may not be successful if the external environmental conditions change rapidly because N, P and K in natural ecosystems respond differently to changes in environmental conditions. N in natural ecosystems mainly originates from biological processes such as atmospheric N fixation and organic matter decomposition. The sources of P and K are mainly controlled by geochemical processes such as rock weathering. Therefore, changes in environmental conditions cause different changes in the availability of soil N, P, and K. Consequently, the plant’s acquisition of N, P, and K will also change, resulting in disproportionate absorption of nutrients, which is another trade-off strategy. Thus, our results suggest that global changes in precipitation and N deposition influence the trade-off strategy for N, P, and K absorption in H. ammodendron. However, it should be highlighted that the trade-off strategy of the disproportionate absorption of N, P, and K is temporary. If precipitation and N input continue to increase for a long time, plants will eventually return to a state of absorbing nutrients proportionately to survive.

The absorption and utilization of elements by plants is an important process in the biogeochemical cycles of terrestrial ecosystems, and it can affect the biogeochemical cycles of elements in terrestrial ecosystems. This study observed that N, P, and K in H. ammodendron and the relationship between N, P, and K changed with N deposition and precipitation, suggesting that the biogeochemical cycles of N, P, and K in desert ecosystems dominated by H. ammodendron would be affected by N deposition and precipitation.

Conclusion

In any water or nitrogen treatment, the WUE remained independent of N, P, and K of H. ammodendron, suggesting that there is no coupling between water use and nutrient economics in the dominant species of the Asian desert. This result was associated with lack of the correlation between N, P, K, and gas exchange in H. ammodendron. However, water addition, N addition, and the interaction between them exerted effects on the relationships between plant N, P, and K. This indicates that global changes in precipitation and atmospheric N deposition will affect the geochemical cycles of N, P, and K in Asian deserts dominated by H. ammodendron, and drive the relationships between plant nutrients to change, thereby causing changes in the trade-off strategy of H. ammodendron.

Supporting tables—Contains all the supporting tables.

(DOCX)

Click here for additional data file.

The N, P, K contents of H. ammodendron.

(XLSX)

Click here for additional data file.

The photosynthetic rate (A), stomatal conductance (gs) and transpiration rate (E) in H. ammodendron.

(XLSX)

Click here for additional data file.

The soil total N (STN), soil total P (STP) and soil Olsen-P (SOP).

(XLSX)

Click here for additional data file.

20 Apr 2021

PONE-D-21-06220

Precipitation change and atmospheric N deposition affect the correlation between N, P and K but not the coupling of water-element in H. ammodondron

PLOS ONE

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Reviewer #1: The manuscript “Precipitation change and atmospheric N deposition affect the correlation between N, P and K but not the coupling of water-element in H. ammodondron” by Chen et al investigated the effects from precipitation change and N deposition on the relationships between different plant nutrient (N, P and K) contents and water-use efficiency of Haloxylon ammodendron, which is a dominant desert plant. The authors showed that addition of water and N altered the correlations between nutrient contents in plant but not the correlations between plant WUE and nutrient contents. This result is interesting because it suggests global change factors such as precipitation change and N deposition can influence terrestrial biogeochemical cycles by affecting plant water-nutrient trade-off strategies. However, the manuscript needs to be properly revised before the acceptance for publication, especially for the language. I will not specify the mistakes in English writing because there were too many. Thus a thorough revision by native English speaker is strongly recommended.

Title: I think the name of the studied plant should be in full spell.

Introduction:

L55 “…, which are affected by plant N…” I don’t quite understand what did this “which” refer to, WUE or gas exchanges? Also for this sentence L55-57 I think the authors should explain further why the WUE (or water use) is expected to be related to plant nutrient contents. This is the basis and significance of understanding the results of this study. I see that the authors tried to bring this issue in L60-62, but the explanation was not clear.

L66-68 Here the authors mentioned that “little attention has been paid to the influence of N deposition and precipitation change on these relationships”. However in the next paragraph, “the effects of rainfall change and N deposition on these couplings have been observed in grassland and forest ecosystems [22,23]” (L77-79). I think the current progresses on this topic should be properly introduced in the section.

Overall this section lacks a clear review on the current knowledge of the coupling between plant WUE and nutrient status (related to absorption and utilization) and its response to global change factors.

Results:

In the experiment there were two factors, that is, water addition and N addition. However, the authors only used ANOVA to analyze the effects of different treatments on plant WUE, nutrients and soil variables. As such, the effects of watering, N addition and their interactions on these variables were not statistically assessed. In addition, the authors used a lot of “slightly” or “slight” in describing the changes in certain variables by treatments of watering or N addition. This is not appropriate because it cannot reflect the significance of the statistical tests. The description of results should be precise about whether the change is significant or not. Words like “slightly higher” should be revised unless the authors feel a strong need to describe the result that way.

L223-225 It is my feeling that this subtitle, as well as the next ones in L252-253 and L273-275 are too long.

L273-275 This subtitle is confusing. Does it means that here the authors want to present the correlations of data from all treatments?

Also the figures and their captions can be improved. I understand that the authors are trying to present the different correlations of N-P (and N-K and P-K) as affected by watering or N addition by comparing the slopes (or significance) of the linear regressions. It would be better to present the linear regression lines in a same plot, which may provide a more direct view on the effect of watering or N addition level. For example in Figure 2, for the N-P relationship, the authors may put data from W0 and W1 treatments all in one plot but with different colors, which may help to show the different N-P relationships as affected by watering level.

Discussions:

L308-321 I feel that it is better to put these sentences in the Introduction section. Some other sentences in this paragraph are also useful in the Introduction as information of the relationship between plant WUE and nutrient status.

L337-345 Here the authors proposed that the lack of significant coupling between WUE and nutrient contents is related to the slow-growing of H. ammodendron plant. I wonder if there is any special characteristic in leaf or root morphology of this species that helps to survive the desert. These characteristics may also be relevant for the uncoupled plant WUE and nutrient status.

L362-366 I would like to see a more precise explanation on the mechanisms of the trade-off strategies that result in the “proportional” and “disproportional” absorption of nutrients by plant.

L394-398 This sentence is too long and confusing.

Overall I feel the authors discussed too many on the changes in plant NPK contents with watering and N addition and used it as the explanation of the varied NPK coupling as affected by watering and N addition. There are many literatures on the ecosystem stoichiometry including plant nutrient ratios. I suggest that the authors should consider the underlying mechanisms of theses plant stoichiometric ratios, and make efforts to explain their results based on both plant physiology and ecology aspects.

Conclusion:

L414-415 “… all exerted significant effects …” From what I understand, this statement is based on comparing the slope of linear regressions (or correlation coefficients) under different watering or N addition levels. I don’t see a statistical analysis on the effect of watering, N addition and their interactions on the correlations. So the word “significant” can be misleading.

Reviewer #2: The manuscript titled: “Precipitation change and atmospheric N deposition affect the correlation between N, P and K but not the coupling of water-element in H. ammodondron” by Chen et al. presents interesting results on the Gurbantunggut Desert. But there are a few significant limitations that need to be addressed in a revised version of this manuscript.

1. The whole paper needs a thorough edit (and spelling check) by an English first-language speaker before resubmission.

2. Results and Discussion: Your paper has two sections such as “Results and discussion”, so you should just represent the results with concise and precise sentences and then take further discussion in the discussion section. But in your paper, lots of discussion sentences appear your results section. Please cancel all information related to discussion in your results section

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: Comments to the Author.docx

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12 May 2021

Reviewer 1

Title: I think the name of the studied plant should be in full spell.

Response:

Thank you for your suggestion! We have revised the title. Please see line 3 at the Revised Manuscript with Track Changes.

Introduction:

L55 “…, which are affected by plant N…” I don’t quite understand what did this “which” refer to, WUE or gas exchanges? Also for this sentence L55-57 I think the authors should explain further why the WUE (or water use) is expected to be related to plant nutrient contents. This is the basis and significance of understanding the results of this study. I see that the authors tried to bring this issue in L60-62, but the explanation was not clear.

Response:

Thank you for your comment! We have revised our writing and added the reason why WUE is expected to be related to plant N, P and K. Please see line 56-68 at the Revised Manuscript with Track Changes.

L66-68 Here the authors mentioned that “little attention has been paid to the influence of N deposition and precipitation change on these relationships”. However in the next paragraph, “the effects of rainfall change and N deposition on these couplings have been observed in grassland and forest ecosystems [22,23]” (L77-79). I think the current progresses on this topic should be properly introduced in the section.

Response:

Sorry! Our writing may mislead you. From line 74 to line 108 at the Revised Manuscript with Track Changes, we first put forward the main concern in our article, which is also the problem that this article will solve. Then around this concern, we made 3 hypotheses. (1) The main concern is that little attention has been paid to the influence of N deposition and precipitation change on these relationships between WUE and plant N, P, K, thus, there is great uncertainty about this issue; although previous studies have reported that N deposition and precipitation change have resulted in the variation of plant N/P ratio and N/K ratio, which indirectly confirmed that plant element coupling was affected by the changes in rainfall and N deposition (see lines 74-89). (2) The coupling between WUE and plant N, P, K and the coupling between plant elements have been found in grassland and forest ecosystems, we made the first hypothesis that these couplings should also occur in H. ammodendron (see lines 96-100). (3) The effects of rainfall change and N deposition on these element couplings have been observed in two meta-analyses, thus, we made the second hypothesis that the correlation between plant nutrients should also vary with changes in rainfall and atmospheric N deposition for H. ammodendron (see lines 100-104). (4) The respond of elements to N deposition and precipitation may vary across elements, and WUE is influenced by N deposition and precipitation; therefore we made the third hypothesis that changes in rainfall and atmospheric N deposition also have an effect on these correlations between WUE and N, P, K (see lines 104-108).

Overall this section lacks a clear review on the current knowledge of the coupling between plant WUE and nutrient status (related to absorption and utilization) and its response to global change factors.

Response:

Thank you for your comment! We have added the review on the current knowledge of the coupling between plant WUE and nutrient status (related to absorption and utilization) and its response to global change factors, especially precipitation and atmospheric N deposition. Please see the Introduction section (Lines 56-68, 80-89 at the Revised Manuscript with Track Changes).

Results:

In the experiment there were two factors, that is, water addition and N addition. However, the authors only used ANOVA to analyze the effects of different treatments on plant WUE, nutrients and soil variables. As such, the effects of watering, N addition and their interactions on these variables were not statistically assessed. In addition, the authors used a lot of “slightly” or “slight” in describing the changes in certain variables by treatments of watering or N addition. This is not appropriate because it cannot reflect the significance of the statistical tests. The description of results should be precise about whether the change is significant or not. Words like “slightly higher” should be revised unless the authors feel a strong need to describe the result that way.

Response:

Thank you for your suggestions. Based on these suggestions, we have made a big change. We have conducted a two-way ANOVA analysis to assess the effects of watering, N addition and their interactions on plant N, P, K and soil N, P. Please see lines 216-233 and Table 1 at the Revised Manuscript with Track Changes. In addition, we have simplified the expression of the results, and deleted many verbose and meaningless expressions.

L223-225 It is my feeling that this subtitle, as well as the next ones in L252-253 and L273-275 are too long.

Response:

Thank you for your comment. We have revised these subtitles. Please see lines 244-246, 273-275 and 297-299 at the Revised Manuscript with Track Changes.

L273-275 This subtitle is confusing. Does it means that here the authors want to present the correlations of data from all treatments?

Response:

Sorry for our poor writing. In this sub-section, we presented the correlation between WUE and N, P, K and the correlation between plant N, P, K under two conditions: adding N and no adding water (W0 + N treatments, includes W0N1 and W0N2) and adding both N and water (W1 + N treatments, includes W1N1 and W1N2). The aim is to assess the effect of the interaction of water and N addition on the correlation between WUE and N, P, K and the correlation between plant N, P, K. We have revised this subtitle. Please see lines 297-299 at the Revised Manuscript with Track Changes.

Also the figures and their captions can be improved. I understand that the authors are trying to present the different correlations of N-P (and N-K and P-K) as affected by watering or N addition by comparing the slopes (or significance) of the linear regressions. It would be better to present the linear regression lines in a same plot, which may provide a more direct view on the effect of watering or N addition level. For example in Figure 2, for the N-P relationship, the authors may put data from W0 and W1 treatments all in one plot but with different colors, which may help to show the different N-P relationships as affected by watering level.

Response:

Thank you for your suggestion. We have revised the figure, please see Fig. 2-4.

Discussions:

L308-321 I feel that it is better to put these sentences in the Introduction section. Some other sentences in this paragraph are also useful in the Introduction as information of the relationship between plant WUE and nutrient status.

Response:

Thank you for your comment. We have moved these sentences into the Introduction. Please see lines 56-68 at the Revised Manuscript with Track Changes.

L337-345 Here the authors proposed that the lack of significant coupling between WUE and nutrient contents is related to the slow-growing of H. ammodendron plant. I wonder if there is any special characteristic in leaf or root morphology of this species that helps to survive the desert. These characteristics may also be relevant for the uncoupled plant WUE and nutrient status.

Response:

Your idea is correct. It may be associated with the roots of H. ammodendron. They are inserted into the soil layer deeper than 3 m to uptake groundwater (Sheng et al., 2004), so groundwater is an important or most important source of water for the plant species. The N, P, K contents in groundwater may be relatively low. Therefore, even if gas exchanges change, the absorption of these elements by H. ammodendron does not necessarily change accordingly, resulting in gas exchanges independent of N, P, K and thus no correlation between WUE and N, P, K. We added the possible mechanism. please see lines 353-360 at the Revised Manuscript with Track Changes.

L362-366 I would like to see a more precise explanation on the mechanisms of the trade-off strategies that result in the “proportional” and “disproportional” absorption of nutrients by plant.

Response:

Thank you for your comment. The proportional absorption is caused by the proportional requirement of N, P, K by plants. The disproportional absorption may be associated with disproportional availabilities of soil N, P, K induced by adding water and adding N. However, the trade-off strategies of disproportional absorption of N, P and K is temporary. If precipitation and N input increase for a long time, in order to survive, plants will eventually return to a state of absorbing nutrients in proportion. We have added the detailed explanation, please see lines 396-407 at the Revised Manuscript with Track Changes.

L394-398 This sentence is too long and confusing.

Response:

Thank you for your comment. Since this sentence is verbose and meaningless, we have deleted it. Furthermore, we have deleted S1 Table and S3 Table because these two tables are also meaningless.

Overall I feel the authors discussed too many on the changes in plant NPK contents with watering and N addition and used it as the explanation of the varied NPK coupling as affected by watering and N addition. There are many literatures on the ecosystem stoichiometry including plant nutrient ratios. I suggest that the authors should consider the underlying mechanisms of theses plant stoichiometric ratios, and make efforts to explain their results based on both plant physiology and ecology aspects.

Response:

Thank you for your suggestion. Based on the suggestion, we made a great revision on the discussion (see lines 374-395 at the Revised Manuscript with Track Changes). We used the knowledges of the physiology of K (lines 376-380) and the ecology theory of P limitation (lines 380-387) to explain our results. In addition, in order to prove that H. ammodendron suffered from P limitation, we have added a new S3 Table showing the N/P ratio in H. ammodendron.

Conclusion:

L414-415 “… all exerted significant effects …” From what I understand, this statement is based on comparing the slope of linear regressions (or correlation coefficients) under different watering or N addition levels. I don’t see a statistical analysis on the effect of watering, N addition and their interactions on the correlations. So the word “significant” can be misleading.

Response:

Thank you for your suggestion. We have deleted this word. Please see line 428 at the Revised Manuscript with Track Changes.

Reviewer 2

1. The whole paper needs a thorough edit (and spelling check) by an English first-language speaker before resubmission.

Response:

Thank you for your suggestion. We have asked Dr. Eric Posmentier in the department of Earth Sciences, Dartmouth College to edit English.

2. Results and Discussion: Your paper has two sections such as “Results and discussion”, so you should just represent the results with concise and precise sentences and then take further discussion in the discussion section. But in your paper, lots of discussion sentences appear your results section. Please cancel all information related to discussion in your results section

Response:

Thank you for your suggestion. we have simplified the expression of the results, and deleted many verbose and meaningless expressions and the discussion in the Results Section. Please see lines 216-233, 255-257, 286-289 and 310-312 at the Revised Manuscript with Track Changes.

3. Specific comments：

Line 49 water is the Plant water economy and nutrition economy? please define it clearly, and early, in the introduction.

Response:

Thank you for your suggestion. We have revised our writing. Please see lines 50-51 at the Revised Manuscript with Track Changes.

Line 55-56 I think references are needed here.

Response:

Thank you for your suggestion. We have added a description of the mechanism behind the correlation between WUE and gas exchange and N, P, K; and references have been cited in this part. Please see lines 56-68 at the Revised Manuscript with Track Changes.

Line 77-81 is there a direct logical relationship between the two ecosysytems?

Response:

Thank you for your comment. We made a mistake in the previous version. These two previous studies [32,33] (the original version is [22, 23]) are meta-analysis on global scale, not research done in a single ecosystem. We have revised the expression, please see lines 100-102 at the Revised Manuscript with Track Changes. Since the effects of changes in rainfall change and N deposition on the N, P, K couplings have been observed on global scale, we assumed that the correlation between plant nutrients should also vary with changes in rainfall and atmospheric N deposition for H. ammodendron. This is the logic of our writing.

Line 106 suggest to revises “woods” to “trees”

Response:

Thank you for your suggestion. We have revised this word. Please see line 133 at the Revised Manuscript with Track Changes.

Line 136 suggest to delete “Due to no leaves,” I think this is an inaccurate statement.

Response:

Thank you for your suggestion. We have revised our writing. Please see line 163 at the Revised Manuscript with Track Changes.

Line 156 suggest to delete “which was its prime assimilating organ”

Response:

Thank you for your suggestion. We have deleted this sentence. Please see line 184 at the Revised Manuscript with Track Changes.

Line 156 what is “slight positive”? It is unclear

Response:

Sorry. I don’t see “slight positive” in line 156. However, due to this expression is unclear and useless, we have deleted this expression in the whole manuscripts.

Line 156 How to understand “no relationships between…….” I think this is an inaccurate statement

Response:

Thank you for your suggestion. We have revised our writing. Please see line 251 at the Revised Manuscript with Track Changes.

Line 264-266 Please, check the language in this statement. It is unclear

Response:

Thank you for your suggestion. Since this sentence may be inapposite. We have deleted it. Please see lines 286-289 at the Revised Manuscript with Track Changes.

Submitted filename: responses to the review-3.docx

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4 Jun 2021

PONE-D-21-06220R1

Changes in Precipitation and atmospheric N deposition affect the correlation between N, P and K but not the coupling of water-element in Haloxylon ammodondron

PLOS ONE

Dear Dr. Wang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Please check your manuscript carefully, especially semicolon. The reviewer  has given details on format mistakes.

==============================

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Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

Please check your manuscript carefully, especially semicolon. The reviewer has give details on format mistakes.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I see that the manuscript was greatly improved from the old version. There are still some problems, but most of them are minor and easy to correct. In particular I suggest the authors to take care of the use of semicolon. There were quite a lot in the manuscript, which made some sentences too long to read.

Here are my specific comments on the ms:

L52: The first appearance of N, P, and K should be in their full spell. In addition, it is better to use their full spell if they are the first words in a sentence, e.g. L61 and L64.

L65: change to “osmotic pressure and enzyme activity [19-21], and thus strongly controls …”

L74-77: The first sentence is the same with L48-49, so it is not necessary to state again. Please delete these texts before the semicolon. In addition, I noticed that the authors used quite a lot of semicolons in the text, which has caused the manuscript to be less readable. Please revise.

L82-84: Here is an example of the bad use of semicolon. The sentence should be separated into two sentences for better reading.

L88-89: Another semicolon again. In addition, I feel that the two parts have stated the similar meaning. The influence of precipitation and N deposition change on WUE-nutrient relationship is less known, so of course there is uncertainty. But what does this uncertainty mean or imply? I suggest the authors should expand further to explain this uncertainty, otherwise the latter sentence after the semicolon is useless and should be deleted.

L96-108: I see that the authors are trying to explain how did these hypotheses come out, but the texts are in fact a repeated of those in the above paragraphs. I suggest to revise these sentences into “Given the universal plant WUE-nutrient coupling and plant NPK coupling, and the possible impact of changes in precipitation and N deposition on these couplings, we hypothesized that: (1)…; (2)…; (3)….”

L108: change to “we conduced a field experiment …”

L112: “grown” should be “growing”

L113: move “in desert ecosystem” to the end of this sentence.

L217: Please check if this is the standard form of unit by PLOS publication.

L218-223: More semicolons. Why use the semicolon when the sentence can be finished with a period? Also, I don’t follow the use of p-value here and the next paragraph with STN and SOP. Does the p-value represent t-test or LSD comparison of two treatments? If so, this should be clearly stated either here or in the statistical analysis method section. There should be only one p-value for the ANOVA with six treatments.

L223-226: The same problem about semicolon. In addition, these p-values are actually presented in the table, so it is not necessary to repeatedly present in the text. The same problem also happened in the following sections of results, please check and revise.

L230: Change to “STP did not change significantly across treatments”, and delete “p>0.05”. Same for the last sentence and elsewhere in the manuscript.

Also, I wonder why the data for WUE under different treatments were not provided. Did WUE change significantly with N addition and watering?

L247-251: Still the semicolon problem. The text clearly presented two sets of the results, i.e. the WUE-NPK correlations and the NPK correlations, so why not separate them with a period? Also details of the linear regressions were provided in the figure and again in the Table 2, and once again in the text here. Please only present what you think the most important information in the parentheses and delete the repeated ones. The same problem with the next sentence and sentences in the next two paragraphs.

L323: change to “there was no significant correlation”

L325-327: This sentence means the same with L322-324. Please delete it.

L341-343: This sentence described a result (Table S2), which should be mentioned in the Results. Overall the results of WUE (or gas exchange parameters) were lost from the Results. Does the authors have a good reason for not presenting these results?

L348: change “and” to “but”

L352: change to “needs confirmation” or “needs to be confirmed”

L353: There is no need to start a new paragraph because here presented another reason for the non-significant correlation between WUE and plant NPK.

L364-370: These three sentences can be simplified into one sentence. For example: “The correlations between plant NPK varied with water addition levels (Fig. 2), N addition levels (Fig. 3), as well as the interaction between N addition and watering (Fig. 4). There results confirm …”

L375: change to “in that case”

L380-381: change “on the other hand” to “in contrast”. Also is there any reference or explanation on this description that plant demands for P is greater than that for N when precipitation increases (the authors did not point out which direction of the change in precipitation)?

L382-384: Bad grammar. Change to “… indicator of the relative limitation of N versus P in ecosystems, i.e., N/P ratio less than 14 generally indicates N limitation while N/P ratio greater than 16 suggests P limitation.”

L384-385: “almost higher than 16”? Do you mean “mostly higher than 16”? From Table S3, all mean N/P ratios were higher than 16. Also this should be mentioned in the Results section.

L394: Where did the “interaction of temperature and precipitation” come from?

Reviewer #2: This is the second time I have reviewed this manuscript, and I feel the authors have really improved the manuscript and limited the scope of their results to accurately reflect what the data are indicating.but,the standard of English is poor - even in this revision. Providing this is done, the manuscript is in my opinion acceptable for publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

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Submitted filename: Comments to the Author2.docx

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21 Jun 2021

Reviewer #1: I see that the manuscript was greatly improved from the old version. There are still some problems, but most of them are minor and easy to correct. In particular I suggest the authors to take care of the use of semicolon. There were quite a lot in the manuscript, which made some sentences too long to read.

Response:

Thank you for your suggestion! We have reduced the use of semicolon.

Here are my specific comments on the ms:

L52: The first appearance of N, P, and K should be in their full spell. In addition, it is better to use their full spell if they are the first words in a sentence, e.g. L61 and L64.

Response:

Thank you for your suggestion! We have revised the spell. Please see lines 54, 55, 57, 65 and 68 at the Revised Manuscript with Track Changes.

L65: change to “osmotic pressure and enzyme activity [19-21], and thus strongly controls …”

Response:

Thank you for your suggestion! We have revised the sentence. Please see lines 69-70 at the Revised Manuscript with Track Changes.

L74-77: The first sentence is the same with L48-49, so it is not necessary to state again. Please delete these texts before the semicolon. In addition, I noticed that the authors used quite a lot of semicolons in the text, which has caused the manuscript to be less readable. Please revise.

Response:

Thank you for your suggestion! We have revised the sentence. Please see lines 79-81 at the Revised Manuscript with Track Changes. And we have reduced the use of semicolon as far as possible.

L82-84: Here is an example of the bad use of semicolon. The sentence should be separated into two sentences for better reading.

Response:

Thank you for your suggestion! We have revised the sentence. Please see lines 88-90 at the Revised Manuscript with Track Changes.

L88-89: Another semicolon again. In addition, I feel that the two parts have stated the similar meaning. The influence of precipitation and N deposition change on WUE-nutrient relationship is less known, so of course there is uncertainty. But what does this uncertainty mean or imply? I suggest the authors should expand further to explain this uncertainty, otherwise the latter sentence after the semicolon is useless and should be deleted.

Response:

Thank you for your suggestion! We have revised the sentence. Please see line 96 at the Revised Manuscript with Track Changes.

L96-108: I see that the authors are trying to explain how did these hypotheses come out, but the texts are in fact a repeated of those in the above paragraphs. I suggest to revise these sentences into “Given the universal plant WUE-nutrient coupling and plant NPK coupling, and the possible impact of changes in precipitation and N deposition on these couplings, we hypothesized that: (1)…; (2)…; (3)….”

Response:

Thank you for your suggestion! We have revised the sentence. Please see lines 104-111 at the Revised Manuscript with Track Changes.

L108: change to “we conduced a field experiment …”

Response:

Thank you for your suggestion! We have revised the sentence. Please see line 122 at the Revised Manuscript with Track Changes.

L112: “grown” should be “growing”

Response:

Thank you for your suggestion! We have revised the word. Please see line 127 at the Revised Manuscript with Track Changes.

L113: move “in desert ecosystem” to the end of this sentence.

Response:

Thank you for your suggestion! We have revised the sentence. Please see lines 128-129 at the Revised Manuscript with Track Changes.

L217: Please check if this is the standard form of unit by PLOS publication.

Response:

Thank you for your suggestion! We have revised the unit. Please see lines 243-244 and 255-256 at the Revised Manuscript with Track Changes.

L218-223: More semicolons. Why use the semicolon when the sentence can be finished with a period? Also, I don’t follow the use of p-value here and the next paragraph with STN and SOP. Does the p-value represent t-test or LSD comparison of two treatments? If so, this should be clearly stated either here or in the statistical analysis method section. There should be only one p-value for the ANOVA with six treatments.

Response:

Thank you for your suggestion! We have deleted the semicolon. Please see lines 244-250 at the Revised Manuscript with Track Changes. The p-value represent LSD comparison, we have added the statements. Please see lines 230-232, 246, 248-250, and 258-260.

L223-226: The same problem about semicolon. In addition, these p-values are actually presented in the table, so it is not necessary to repeatedly present in the text. The same problem also happened in the following sections of results, please check and revise.

Response:

Thank you for your suggestion! We have deleted the semicolon and the redundant p-values. Please see lines 250-254 at the Revised Manuscript with Track Changes.

L230: Change to “STP did not change significantly across treatments”, and delete “p>0.05”. Same for the last sentence and elsewhere in the manuscript.

Response:

Thank you for your suggestion! We have revised the sentence. Please see lines 258-259 and 262 at the Revised Manuscript with Track Changes.

Also, I wonder why the data for WUE under different treatments were not provided. Did WUE change significantly with N addition and watering?

Response:

The data have been published in another paper (Chen et al., 2021, Biogeosciences, 18, 2859–2870). In addition, the main objective of the present study is to determine the effect of precipitation and atmospheric N deposition on the correlation between N, P and K and between WUE and N, P, K in H. ammodendron. Therefore, the data for WUE under different treatments has not been provided in this manuscript. We have deleted the description of WUE and gas exchange measurement in the Materials and methods Section, and added a statement about the data source of WUE and gas exchange in the manuscript. Please see lines 236-238 at the Revised Manuscript with Track Changes. We have reported in the paper published in BG that WUE changed significantly with N addition and watering.

L247-251: Still the semicolon problem. The text clearly presented two sets of the results, i.e. the WUE-NPK correlations and the NPK correlations, so why not separate them with a period? Also details of the linear regressions were provided in the figure and again in the Table 2, and once again in the text here. Please only present what you think the most important information in the parentheses and delete the repeated ones. The same problem with the next sentence and sentences in the next two paragraphs.

Response:

Thank you for your suggestion! We have revised the sentence and deleted the redundant information in the parentheses. Please see lines 275-283 at the Revised Manuscript with Track Changes.

L323: change to “there was no significant correlation”

Response:

Thank you for your suggestion! We have revised the sentence. Please see line 337 at the Revised Manuscript with Track Changes.

L325-327: This sentence means the same with L322-324. Please delete it.

Response:

Thank you for your suggestion! We have deleted the sentence. Please see line 340-341 at the Revised Manuscript with Track Changes.

L341-343: This sentence described a result (Table S2), which should be mentioned in the Results. Overall the results of WUE (or gas exchange parameters) were lost from the Results. Does the authors have a good reason for not presenting these results?

Response:

Thank you for your suggestion! However, the aim of the present study is to determine the effect of precipitation and atmospheric N deposition on the correlation between N, P and K and between WUE and N, P, K in H. ammodondron. In addition, the results of gas exchanges and WUE have been reported in our paper published in Biogeosciences (Chen et al., 2021, Biogeosciences, 18, 2859–2870). We presented the results of the correlation between N, P, K and gas exchange in S2 Tables to only explain the observed irrelevance between N, P, K and WUE. So we did not present them in the Results section.

L348: change “and” to “but”

Response:

Thank you for your suggestion! We have revised the word. Please see line 364 at the Revised Manuscript with Track Changes.

L352: change to “needs confirmation” or “needs to be confirmed”

Response:

Thank you for your suggestion! We have revised the sentence. Please see lines 367-368 at the Revised Manuscript with Track Changes.

L353: There is no need to start a new paragraph because here presented another reason for the non-significant correlation between WUE and plant NPK.

Response:

Thank you for your suggestion! We have merged the two paragraphs. Please see line 368 at the Revised Manuscript with Track Changes.

L364-370: These three sentences can be simplified into one sentence. For example: “The correlations between plant NPK varied with water addition levels (Fig. 2), N addition levels (Fig. 3), as well as the interaction between N addition and watering (Fig. 4). There results confirm …”

Response:

Thank you for your suggestion! We have revised the sentence. Please see lines 380-390 at the Revised Manuscript with Track Changes.

L375: change to “in that case”

Response:

Thank you for your suggestion! We have revised the sentence. Please see line 392 at the Revised Manuscript with Track Changes.

L380-381: change “on the other hand” to “in contrast”. Also is there any reference or explanation on this description that plant demands for P is greater than that for N when precipitation increases (the authors did not point out which direction of the change in precipitation)?

Response:

Thank you for your suggestion! We have revised the sentence. Please see line 397 at the Revised Manuscript with Track Changes. We made some mistakes in this description. What we want to express is that H. ammodendron in the present study may demand more P than N under precipitation increase. The reason is that the N/P ratio in H. ammodendron was mostly higher than 16, which have been mentioned later (lines 366-371). We have revised this description. Please see lines 403-404 at the Revised Manuscript with Track Changes.

L382-384: Bad grammar. Change to “… indicator of the relative limitation of N versus P in ecosystems, i.e., N/P ratio less than 14 generally indicates N limitation while N/P ratio greater than 16 suggests P limitation.”

Response:

Thank you for your suggestion! We have revised the sentence. Please see lines 399-402 at the Revised Manuscript with Track Changes.

L384-385: “almost higher than 16”? Do you mean “mostly higher than 16”? From Table S3, all mean N/P ratios were higher than 16. Also this should be mentioned in the Results section.

Response:

Thank you for your suggestion! We have change “almost” to “mostly”. Please see line 403 at the Revised Manuscript with Track Changes. Also, the result of N/P ratio mentioned here is to explain that the demand for P is higher than the demand for N in H. ammodendron in the present study, not the key results of the present study. So we did not present it in the Results section.

L394: Where did the “interaction of temperature and precipitation” come from?

Response:

Sorry for our mistake. This sentence should be “interaction of N deposition and precipitation”. We have revised it. Please see line 414 at the Revised Manuscript with Track Changes.

Reviewer #2: This is the second time I have reviewed this manuscript, and I feel the authors have really improved the manuscript and limited the scope of their results to accurately reflect what the data are indicating. but, the standard of English is poor - even in this revision. Providing this is done, the manuscript is in my opinion acceptable for publication.

Response:

Thank you for your suggestion. We have asked an English editing company (Editage) to edit English.

Submitted filename: Responses to the Reviewers.docx

Click here for additional data file.

21 Jul 2021

PONE-D-21-06220R2

Changes in precipitation and atmospheric N deposition affect the correlation between N, P and K but not the coupling of water-element in Haloxylon ammodendron

PLOS ONE

Dear Dr. Wang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Please pay attention to the citation of equations in Discussion. You have deleted them in Materials and methods.

The format of Reference need to be checked carefully.

==============================

Please submit your revised manuscript by Sep 04 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Yajuan Zhu, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

The author had deleted Measurements of gas exchanges and calculations of water use efficiency in Materials and method. However, in L282, there are citations of two equations in Discussion: "According to the definitions of ins-WUE and int-WUE (equations (1) and (2))," Please modify discussion according to Materials and method.

The full comma "." was missing in Line 332.

There are some format mistakes in References. In 21, 22, 25, 37, 39, 50, 54 and 55, all Latin name in title should be italic. In 22, genus name should be Artemisia and Caragana. In 50, only the first letter of the first word need to be capital. Please check them carefully.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript was carefully revised based on the comments raised by both reviewers, and the writing has been greatly improved. I feel that this ms is now suitable for publication in PLOS One.

Reviewer #2: (No Response)

**********

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

6 Aug 2021

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Responses:

Thank you for your suggestion. We have reviewed our reference list. We have not cited any retracted papers. In the last revision, we have added 1 reference, it is reference [46]. However, we did forget to mention this change in the Responses to the Reviewers of the previous version. Sorry for our mistake in the last revision.

Additional Editor Comments (if provided):

The author had deleted Measurements of gas exchanges and calculations of water use efficiency in Materials and method. However, in L282, there are citations of two equations in Discussion: "According to the definitions of ins-WUE and int-WUE (equations (1) and (2))," Please modify discussion according to Materials and method.

Responses:

Thank you for your suggestion. We have revised the sentence. Please see lines 281-285 at the Revised Manuscript with Track Changes.

The full comma "." was missing in Line 332.

Responses:

Thank you for your suggestion. We have added the full comma “.”. Please see line 334 at the Revised Manuscript with Track Changes.

There are some format mistakes in References. In 21, 22, 25, 37, 39, 50, 54 and 55, all Latin name in title should be italic. In 22, genus name should be Artemisia and Caragana. In 50, only the first letter of the first word need to be capital. Please check them carefully.

Responses:

Thank you for your suggestion. We have checked the References and revised the mistake. Please see the References section at the Revised Manuscript with Track Changes.

Submitted filename: Responses to the Reviewers.docx

Click here for additional data file.

10 Sep 2021

PONE-D-21-06220R3Changes in precipitation and atmospheric N deposition affect the correlation between N, P and K but not the coupling of water-element in Haloxylon ammodendronPLOS ONE

Dear Dr. Wang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Please pay attention to the citation of equations in Discussion. You have deleted them in Materials and methods.

The format of Reference need to be checked carefully.

==============================

Please submit your revised manuscript by Oct 25 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Yajuan Zhu, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

Please pay attention to the citation of equations in Discussion. You have deleted them in Materials and methods.

The format of Reference need to be checked carefully.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

7 Oct 2021

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Responses:

Thank you for your suggestion. We have reviewed our reference list. We have not cited any retracted papers. In the last revision, we have added 1 reference, it is reference [46]. However, we did forget to mention this change in the Responses to the Reviewers of the previous version. Sorry for our mistake in the last revision.

Additional Editor Comments (if provided):

The author had deleted Measurements of gas exchanges and calculations of water use efficiency in Materials and method. However, in L282, there are citations of two equations in Discussion: "According to the definitions of ins-WUE and int-WUE (equations (1) and (2))," Please modify discussion according to Materials and method.

Responses:

Thank you for your suggestion. We have revised the sentence. Please see lines 281-285 at the Revised Manuscript with Track Changes.

The full comma "." was missing in Line 332.

Responses:

Thank you for your suggestion. We have added the full comma “.”. Please see line 334 at the Revised Manuscript with Track Changes.

There are some format mistakes in References. In 21, 22, 25, 37, 39, 50, 54 and 55, all Latin name in title should be italic. In 22, genus name should be Artemisia and Caragana. In 50, only the first letter of the first word need to be capital. Please check them carefully.

Responses:

Thank you for your suggestion. We have checked the References and revised the mistake. Please see the References section at the Revised Manuscript with Track Changes.

Submitted filename: Responses to the Reviewers.docx

Click here for additional data file.

11 Oct 2021

Changes in precipitation and atmospheric N deposition affect the correlation between N, P and K but not the coupling of water-element in Haloxylon ammodendron

PONE-D-21-06220R4

Dear Dr. Wang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Yajuan Zhu, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have modified all questions and format mistakes. Now it's ready for publish.

Reviewers' comments:

15 Oct 2021

PONE-D-21-06220R4

Changes in precipitation and atmospheric N deposition affect the correlation between N, P and K but not the coupling of water-element in Haloxylon ammodendron

Dear Dr. Wang:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Yajuan Zhu

Academic Editor

PLOS ONE