Ecophysiological responses to drought stress in Populus euphratica

ChunYan Zhao,JianHua Si*,Qi Feng,TengFei Yu,Huan Luo,Jie Qin

1.Northwest Institute of Eco-Environment and Resources,Chinese Academy of Sciences,Lanzhou,Gansu 730000,China

2.Key Laboratory of Ecohydrology of Inland River Basin,Chinese Academy of Sciences,Lanzhou,Gansu 730000,China

3.University of Chinese Academy of Sciences,Beijing 100049,China

ABSTRACT Ecophysiological responses to drought stress of Populus euphratica in Alashan Desert Eco-hydrology Experimental Research Station were investigated. Results show that under mild and moderate drought stress, stomatal length, aperture,area and density is likely to decrease in the early days,but afterwards this is likely to recovery with treatment over the pas‐sage of treatment time. Under severe drought stress, these properties appear to decline continuously. However, after 45 days of drought-stress treatment, the decline is not as noticeable as before, indicating that Populus euphratica could possibly reduce water evaporation by shutting down the stoma,leading to an improvement in its water use efficiency with better survival under drought stress conditions. The leaf area first decreases, and then increases under mild and moderate drought stress conditions, with the average values under different degree of stress found to be approximately 129.52,120.08, 116.63 and 107.28 cm2, respectively. Under moderate stress conditions, the leaf water potential appears to show a continuous decline where the average values under different degree of stress are found to be -1.27, -1.85, -4.29 and-4.80 MPa,respectively.In terms of proline content,the results demonstrate that this factor appears to increase significant‐ly under moderate and severe drought stress conditions. Especially under severe drought stress condition, the content is found to be more than 700 μg/g. Ranging over average values of 14.64 and 15.90 nmol/g under moderate and severe drought stress, respectively, Malondialdehyde content is found to increase quite rapidly under moderate and severe drought stress conditions at first,which then appears to decrease gradually with the treatment over time.

Keywords:stomatal morphology;drought stress;Malondialdehyde;proline;Populus euphratica

1 Introduction

Drought conditions fosters a disadvantaged envi‐ronment that seriously affects plant growth and devel‐opment, especially in arid and semiarid regions (Prad‐han, 2012). Losses in terms of the respective plant's productivity caused by drought stress is nearly equal to total losses induced by all other biotic and abiotic stresses (Barnabaset al., 2008). In order to adapt to drought stress caused by water deficits, plants have developed a number of defense measures. Operational mechanisms of stomata as the potential gateway for modulating water balance and for controlling gas ex‐change play a significant role in the plant's adaptation to drought stress (Backley, 2005; Devireddyet al.,2019).Previous studies have suggested that the stoma‐tal pore area decreases quickly in the face of severe drought stress (Liet al., 2004). Similarly, pronounced decrease in stomatal length and stomatal aperture were also observed in drought stressed plants (Iwaietal., 2003). However, previous studies have focused on the stomatal response in a short period of time after the drought stress treatment. Therefore, further investiga‐tion of the stomatal morphology regulation over a lon‐ger period after drought stress treatment is warranted.

Leaf area is also an important adaptive measure for plants to defend against drought stress.Many stud‐ies have reported that the leaf area decreases under certain drought stress, and this inhibition is likely in accord to the degree and duration of drought stress(Gomez-Del-Campoet al., 2003; Massonnetet al.,2015). In a study based on oilseed rape, results show that leaf area decreased with an increase in the degree of drought stress(Jensenet al.,1996).However,some authors also found that there was no significant de‐crease in the value of leaf area in the seedlings investi‐gated under mild drought stress (Anyiaet al., 2004).Understanding how leaf area is able to respond to dif‐ferent degrees of drought stress is critical for evaluat‐ing the various adaptive mechanisms under different stress environments. Moreover, the leaf water poten‐tial is also a direct index that is able to reflect water conditions or water deficit of various plants.It is note‐worthy that this property can easily be utilised to in‐vestigate the drought defense capacity and drought stress degree in different plants (Maréchauxet al.,2015). In a recent study, the soil water content was found to be the primary factor affecting the magni‐tude of the leaf water potential (Dasguptaet al.,2015).A range of other investigations have also indi‐cated that there was an obvious positive correlation between soil water content and leaf water potential(Berovaet al., 2012). On a similar point, the research of Jongdeeet al. (2002) shows that the leaf water po‐tential in fact declined quite substantially under se‐vere drought stress conditions. However, previous studies on the effects of drought stress on leaf water potential has primarily focused on the qualitative de‐scriptions whereas its quantitative outcomes is still quite scarce in published literature.

As a significant osmotic adjustment chemical sub‐stances,the amount of proline in any plant plays a piv‐otal role in the protection of plants against damage caused by drought conditions. Several authors have observed that the proline content in leaves exhibits an increasing trend under drought stress (Heidariet al.,2009;Hanifet al.,2020).Similar results were also ob‐tained for the case of barley(Singhet al.,1972).How‐ever, contrary to previous results, recent studies have shown that content of proline appears to decrease at first, and then increase in short-term drought stress(Iqbalet al., 2016). There are also some studies that indicate that the proline content of seedling can in‐crease with an increase in the degree of drought stress and the stress treatment time (Manet al., 2011).Therefore, it is particularly important to further ex‐plore the responses of proline to drought stress during a long period of stress conditions, which has not been performed so far. Literature evidence also shows that Malondialdehyde (MDA) is a crucial biomarker that is likely to be changed under different drought stress conditions (Baillyet al., 1996). Consequently, MDA is often used to evaluate the oxidative damage of dif‐ferent plants under drought stress.Despite these inves‐tigations, there exist differing perspectives regarding the relationship between MDA and the content of soil water where some studies have demonstrated that the content of leaf MDA was much higher under drought stress (Soleimanzadehet al., 2010). In other studies,one could perceive that there was a negative correla‐tion between MDA and the content of soil water(Zhaiet al., 2017). Therefore, further investigation is need‐ed to determine the precise functions of MDA content under different drought stress conditions.

Populus euphratica(P. euphratica) is a typical species of desert riparian forest with a strong drought-resistant capacity and a high salt-tolerant ability, grown in the lower reaches of Heihe River of Northwest China. This species plays a positive role in developing the social economy, decreasing wind speed, restoring vegetation, regulating climate and maintaining ecological stability in arid and desert ar‐eas. This plant mainly utilizes groundwater and shal‐low soil water to satisfy its growth needs. Investiga‐tions show thatP. euphraticais grown under an envi‐ronment that has a mean annual evaporation amount more than 90 times the amount of precipitation in the same region (Siet al., 2008). A better understanding of the drought's adaptive mechanisms forP. euphrati‐cais a crucial premise for a critical evaluation of the drought tolerance level. However, the defense mea‐sures ofP. euphraticato drought conditions still re‐main poorly understood.

To seek answers to the aforementioned question,the aim of this study is to explore the regulatory mech‐anism of various properties, with particular focus on stomatal morphology, leaf area, leaf water potential,proline and MDA content under drought stress condi‐tions in a riparian desert forest. The aim of this re‐search is to test and validate the following hypothe‐ses: (1) stomatal morphology regulation can reduce the potential damage caused by drought stress onP.euphratica, and (2)P. euphraticacan reduce the dam‐age of drought stress by reducing leaf area,decreasing leaf water potential and accumulating the content of MDA and proline.

2 Materials and method

2.1 Site description

The experimental site(42°01′N,100°21′E)was lo‐cated in Alashan Desert Eco-hydrology Experimental Research Station (Alxa Station) at the inner Mongolia Autonomous Region of Northwest China. The area within this site is characterized as a typical continen‐tal arid climate. The average annual precipitation is about 38 mm, and approximately 75% of all rainfall occurs between the months of June and September.The average annual temperature is about 8.2 °C and the mean annual evaporation exceeds 3,390 mm. In this region,the average annual wind speed(WS)is ap‐proximately 3.4 to 4.0 m/s and the soil type is derived from fluvial sediments (Siet al., 2008). In the study area,P. euphraticais grown as the most dominant plant species since the conditions are highly condu‐cive to its survival in this climatic region.

2.2 Materials

100 seedlings ofP. euphraticaall having the same size from the Ejinaqi region of the Forest Farm were transplanted (in 2014). These were divided into plas‐tic pots (38 cm in height, 38 cm in width, 45 cm in length).In all pots,total soil salt,potassium,phospho‐rus and nitrogen, plus organic matter, were 2.5%,1.52%, 0.06%, 0.10%, 2.52%, respectively. The soil PH was 8.76.Two years later,a total of 48P.euphrati‐caseedlings having similar size and were determined to be generally straight, healthy, and well-grown were selected for the experimental phase.The average mag‐nitude of seedling height was 52.47±2.47 cm and the average ground diameter was 2.12±0.31 cm.The seed‐lings ofP. euphraticawere then divided into four groups, including a non-drought stress group (ND,60%?70% of field capacity), a mild drought stress group (LD, 50%?60% of field capacity), a moderate drought stress group (MD, 40%?50% of field capaci‐ty) and a severe drought stress group (SD, 30%?40%of field capacity).We thus utilized the weighing meth‐od to ensure the soil water content was within the cor‐responding drought stress range. Each experiment group contained twelveP. euphraticaseedlings,which were measured and sampled at an interval of five days from May 8 to July 3,2016.

2.3 Methods

2.3.1 Stomatal morphology measurements

To perform stomatal morphology measurements,the leaves were excised,sealed in vials,and kept cool.This followed the peeling of all the epidermis used the sticking and tearing methods from the fully ex‐panded leaves where 0.2 cm2leaf pieces were taken and examined at 200x magnification level with a mi‐croscope (Olympus CX31, Olympus America Inc.,Center Valley,PA,USA)as shown in Figure 1.Stoma‐tal length (measured in μm), stomatal aperture (mea‐sured in μm) and stomatal area (measured in μm2)were determined by the Motic Images Advanced 3.2 image processing software. The stomatal density(measured in stomata/mm2) was counted through the number of stomata per mm2of the area. Each treat‐ment comprised of six repeats, and for each sample,six visual fields were counted.

Figure 1 An example for stomata of P.euphratica under different degrees of drought stress(ND=no drought stress;LD=mild drought stress;MD=moderate drought stress;SD=severe drought stress)

2.3.2 Leaf area and leaf water potential measurements

The leaf area was measured by scanning the leaves using the Canon Scan Lide 90 (Canon USA Inc.,NY,USA)and then analyzed in terms of the im‐ages produced by the Motic Images Advanced 3.2 image processing software. These measurements were performed at around 11:00. Next, the leaf water potential was also measured with a portable pressure chamber instrument (Model 1505D, Pressure Cham‐ber Instrument, PMS Instrument Company, USA),and the measurement time of this property was 07:00.

2.3.3 MDA measurements

MDA content ofP. euphraticawas also continu‐ously measured over a five day interval after the drought stress treatments were applied.About 1 g of fresh leaves was homogenized in 0.05 mol/L trichlo‐roacetic acid, and then theP. euphraticahomogenate was centrifuged at 5,200 rpm for a total time of 15 minutes. Then, 3.0 mL of supernatant was incu‐bated with 2.5 mL of thiobarbituric acid at about 95°C for a total time of 20 minutes and finally, a spectro‐photometer (DU-70Spectrophotometer, USA) was utilized to measure the absorbance of the supernatant at 600 and 532 nm wave lengths. The content of MDA was thus calculated based on the following formula(Li,2000):

whereCMDAis the content of MAD;OD532,OD600is ab‐sorbance under 532 and 600 nm; A is the overall amount of reactive solution (mL);Vis the overall amount of extract solution (mL);ais the amount of extract solution used to measure (mL) andWis the weight of fresh leaves(g).

2.3.4 Proline measurements

In this study, the content of proline was measured according to the description of Bateset al.(1973).Ap‐proximately 0.2 g of fresh leaves was homogenized in 5.0 mL (3.0%) sulphosalicylic acid, and then 2.0 mL ofP. euphraticahomogenate was reacted with 2.0 mL of glacial acetic acid and 2.0 mL acid ninhydrin for a total time of 30 minutes at a temperature of 100 °C.The reactive solution was then mixed with 4.0 mL of toluene and centrifuged at 3,000 rpm for 10 minutes.Finally, the optical density of the chromophore con‐taining toluene was measured at 520 nm with a spec‐trophotometer (DU-70Spectrophotometer, USA). The content of proline was calculated based on the follow‐ing formula(Li,2000):

whereCprolineis the content of proline (μg/g);Cis the amount of proline determined by a standard curve(μg);Vis the overall amount of extract solution (mL);ais the amount of extract solution used to measure(mL)andVis the weight of the fresh leaves(g).

2.3.5 Meteorological conditions

Meteorological conditions during 2016 were al‐so measured simultaneously with actual plant data via a standard automatic weather station (Meteoda‐ta 3000, Geóniba SA, Madrkd, Spain) located at approximately 100 m from the experimental plot.Measured variables included the values of relative humidity (RH, %), air temperature (Ta, °C), precip‐itation (P, mm) and wind speed (WS, m/s). All en‐vironmental data were recorded via a Zeno3200-AD data logger. Vapor pressure deficit (VPD, kPa)was also determined by the measured values of Taand RH.

2.3.6 Statistical analysis

All data attained on stomatal morphology(i.e.,sto‐matal length,aperture,area and density),leaf area and leaf water potential, MDA content and proline content measurements were represented as means ± standard deviation(M±SD;n=6).The design is completely ran‐domized design (CRD). Statistical significance of the results was performed using analysis of variance(ANOVA)approach using SPSS(IBM,Inc.)19.0 soft‐ware. The linear functions were fitted using Origin 8.0 and the differences were evaluated at a signifi‐cance level ofp=0.05.

3 Results

3.1 Meteorological conditions

Meteorological variables monitored during the experimental period are presented in Figure 2 where the temporal changes in Ta, RH,VPD and WS reveal a significant degree of fluctuation in the overall trend of the data. The daily average values of Ta,RH, VPD and WS, respectively, are found to be about 21.79 °C, 25.42%, 2.11 kPa and 3.65 m/s. The rainfall, however, was 0 mm during the experimental period, indicating thatP. euphraticagrew under ap‐propriate meteorological conditions during the exper‐imental phase.

Figure 2 Temporal changes in environmental variables during the experimental phase in 2016(daily means±standard deviation)

3.2 Regulation of stomatal morphology under drought stress

The regulation of stomatal morphology under drought stress is presented in Figure 3 where the tem‐poral changes inP. euphratica's stomatal length, aper‐ture, area and density under different degrees of drought stress are illustrated. It is evident that under non-drought stress conditions, stomatal length, aper‐ture, area and density increased continuously with the applied treatment over time,and the average valuesat‐tained were about 13.46 μm, 5.10 μm, 30.93 μm2and 169.41 stomata/mm2, respectively. Under mild drought stress conditions, stomatal length, aperture, area and density show a slight decrease in the early days, but afterwards this increased gradually with the treatment time, attaining average values of about 12.51 μm,3.84 μm, 24.11 μm2and 161.42 stomata/mm2, respec‐tively. Under moderate drought stress conditions, how‐ever,stomatal length,aperture,area and density show a continuous decline. In spite of this, those values began to recover after a period of 45 days of drought stress treatment. The average valuesfor these were about 12.44 μm, 3.31 μm, 20.24 μm2and 130.53 stomata/mm2,respectively. It is interesting to note that under severe drought stress conditions, stomatal length, aperture,area and density show a continuous decline.However,after 45 days of drought stress treatment, the decline of those values were not as noticeable as before, lead‐ing to average values of about 12.22 μm, 2.90 μm,16.36 μm2and 130.28 stomata/mm2,respectively.

Figure 3 Changes in P.euphratica stomatal,aperture,area and density under different degrees of drought stress(ND=no drought stress;LD=mild drought stress;MD=moderate drought stress;SD=severe drought stress)

3.3 Regulation of leaf area and leaf water potential under drought stress

The moderating effect on leaf area and leaf water potential ofP. euphraticaduring the experimental phase is presented in Figure 4. Evidently, leaf area shows a continuous increase under non-drought stress conditions.In contrast,under mild drought stress condi‐tions, leaf area revealed a slight decrease especially in the early days,but then this property increased gradual‐ly with the treatment time. Under moderate drought stress conditions, however, leaf area shows a continu‐ous decline but after 45 days of stress treatment, the leaf area began to increase again. It is noteworthy that under severe drought stress conditions, leaf area re‐vealed a continuous decline, which after 50 days stress treatment was not as noticeable as before. The average values of this property under different de‐grees of drought stress were found to be about 129.52,120.08, 116.63 and 107.28 cm2, respectively.When the leaf water potential was examined, this property shows a good degree of fluctuation trend under nondrought stress conditions. However, the leaf water po‐tential registered a slight decrease in the early days under mild drought stress conditions, but the extent of fluctuations increased with the treatment time. The present study shows that under moderate drought stress conditions,the leaf water potential exhibited a continu‐ous decline. However, it is interesting to note that after 40 days of stress treatment, the leaf water potential be‐gan to increase. Notwithstanding this, under severe drought stress conditions, the leaf water potential shows a continuous decline but after 45 days of stress treatment, the decline was not noticeable. Under dif‐ferent drought stress conditions, the average values were-1.27,-1.85,-4.29 and-4.80 MPa,respectively.

Figure 4 Changes in leaf area and leaf water potential of P.euphratica during the experimental period(ND=no drought stress;LD=mild drought stress;MD=moderate drought stress;SD=severe drought stress)

3.4 Regulation of proline and MDA content under drought stress

The obtained results during the experimental phase shows that changes in MDA and proline con‐tent ofP.euphratica(Figure 5)was not noticeable un‐der non-drought stress as well as mild drought stress conditions. However, under moderate and severe drought stress conditions, the content of proline was seen to increase. It was especially true that under se‐vere drought stress conditions,proline content at the fi‐nal stage of the experiment was greater than 700 μg/g and that the MDA content shows a significant degree of fluctuation under non-drought stress and mild drought stress conditions. However, MDA content ap‐peared to increase under moderate and severe drought stress conditions at first, but then it appeared to de‐crease gradually with the treatment time. The experi‐mental results of MDA content shows that the average values were about 14.64 and 15.90 nmol/g under mod‐erate and severe drought stress conditions,respectively.

3.5 The response relationship under different degrees of drought stress

We investigated the response relationship between soil water content and stomatal length, aperture, area and density,leaf area,leaf water potential,proline and MDA content ofP.euphratica(Figure 6).It is evident that the linear fit relationships have significant differ‐ences under different degrees of drought stress (withp<0.05). In fact, the relationship between soil water content and stomatal aperture, stomatal area, leaf wa‐ter potential, and proline content appeared to be tight‐er under severe drought stress conditions. This yield‐edR2values of about 0.72,0.59,0.75 and 0.66,respec‐tively. The relationship between soil water content and stomatal density, and MDA content were closely linked under moderate drought stress conditions withR2of 0.69 and 0.52, respectively. However, the rela‐tionship between soil water content and stomatal length was closely related under mild drought stress conditions withR2=0.47. In terms of the relationship between soil water content and leaf area, we found that under non-drought stress conditions theR2value was 0.48. These results were all statistically signifi‐cant atp<0.05. In addition, we also found a weaker relationship between soil water content and leaf area,MDA content and stomatal density under mild drought stress conditions.

Figure 5 Change in malondialdehyde(MDA)and proline content of P.euphratica during the experimental period(ND=no drought stress;LD=mild drought stress;MD=moderate drought stress;SD=severe drought stress)

Figure 6 The response relationship between soil water content and stomatal length,aperture,area and density,leaf area,leaf water potential,proline content and MDA content of P.euphratica(ND=no drought stress;LD=mild drought stress;MD=moderate drought stress;SD=severe drought stress)

4 Discussions

The speciesP. euphraticahas a series of measures to defend against drought stress conditions through physiological and metabolic regulatory processes de‐veloped during the long-term processes of biological evolution.Stomata morphology regulation,on the oth‐er hand,plays an essential role in such regulatory pro‐cesses. Based on experimental data, our study shows that under mild and moderate drought stress condi‐tions, stomatal length, aperture, area and density ofP.euphraticadecreased in the early days, while exhibit‐ing recovery with the treatment time. Consistent with our results, previous investigations have also shown that stomatal length, aperture, area and density only decreased in the early days(Frankset al.,2001;Inam‐ullahet al., 2005). This notion suggests that stomatal length, aperture, area and density may be subjected limited under moderate drought stress conditions,but these plants are likely to adapt to a stress environ‐ment and consequent recovery with the application of treatments for a prolonged period of time. Howev‐er, under severe stress conditions, stomatal length,aperture, area and density are likely to decline con‐tinuously, and the rate of declining was not notice‐able in the present study with the prolongation of the treatment time. A primary reason for this could be thatP.euphraticacan reduce water evaporation by shutting down the stoma, which could help to im‐prove water use efficiency and survive better under drought stress conditions (Christopheret al., 2015;Esmailpouret al.,2016).Considering this,it is a rea‐sonably good way to reduce water consumption through adjusting stomata morphology and to reduce the damage ofP. euphraticaunder drought stress conditions.

There is no doubt that leaves are an important plant organ in the exchange of energy and matter with the environment which likely to indicate the growth conditions and photosynthetic capacity of the respec‐tive plant under drought stress conditions. Our results have shown that under mild and moderate drought stress conditions, the leaf area ofP. euphraticaap‐peared to continuously decline until the late stage of the stress treatment. Moreover, some leaves actually turned yellow, withered and even fell under severe drought stress conditions and this phenomenon was more obvious on the lateral shoots ofP.euphratica(data not show). These results concur with the con‐clusion that the plant was able to cope with drought stress by the branch die-back process (Roodet al.,2000). This was largely in part due to the fact that the decrease in leaf area is likely to increase water use efficiency when the soil water supply is defi‐cient. However, contrary to our results, the work of Johnsonet al. (2016) pointed out that plants are like‐ly to protect basal organs by sacrificing their distal portions. In light of the present analysis, we believe that a possible reason for this difference is due to the fact that different plants have different leaves that act to regulate strategies under drought stress condi‐tions.Further in-depth studies are thus required to in‐vestigate the difference of leaf tolerance of various plants which grow under various plant communities and environments.

Leaf water potential as a direct index can be also used to indicate the water conditions and the drought stress degree of different plants (Grzesiaket al.,2006). Our result show that under moderate and se‐vere drought stress conditions, the leaf water potential ofP. euphraticadeclined to a significant degree. This reduction should mainly be attributed to the decrease of soil water potential at the surface of the roots.Moreover, it is reasonable to conclude that leaf water potentials may have decreased, leading to an increase in the capability of absorbing water in a passive man‐ner, thus increasing the ability of plants to develop drought resistance. However, the apparent decline in leaf water potential was not noticeable in the late stage of the stress treatment in this study. Clearly, this suggests thatP. euphraticais likely to respond and adapt to these environmental challenges when the plant is subjected to drought stress over a long period of time.

As one of the most important osmotic adjustment substances, proline has a significant relationship with drought resistance of different plants (Yousifiet al.,2010). Our results show that the proline content ofP.euphraticaappeared to increase quite significantly un‐der both the moderate drought stress and severe drought stress conditions. This result is consistent with one study showing that proline accumulation is associated with the drought tolerance of the upland rice varieties (Lumet al., 2014). The increase of pro‐line content, however, can improve the drought toler‐ance of plants due to the osmoregulatory property of proline, and thus can maintain osmotic balance be‐tween the protoplasm and the environment to prevent the dehydration effect on the underlying plant. More‐over,the bond between proline and protein is likely to enhance the hydration of proteins, thus protecting the stability of the structure and function of the macro‐molecules(Pan,2013).

According to the literature,MDA is a crucial prod‐uct of membrane lipid peroxide (Guoet al., 2018). In this study, we found that MDA content ofP. euphrati‐caincreased quickly under moderate drought stress and severe drought stress conditions at first, but then it also decreased gradually with the treatment time.A primary reason for this may be the fact that the rela‐tive permeability of the various cell membranes was subjected to damage in the early days of the drought stress, but permeability is likely to recover with the treatment time (Liuet al., 2016). Indeed, the study of Zhanget al. (2012) noted that the greater the content of MDA, the larger was the damage caused to the cell membranes. In our experiment, we found that the highest value was not attained at the end of stress treatment, but rather this appeared at the moderate term of stress treatment. It is thus possible that the cell membrane ofP.euphraticamay be subjected to serious damage at the first instance, but this damage is likely to be reduced over time andP.euphraticasis likely to adapt to drought stress environments by itself.

The response relationship between soil water con‐tent and stomatal length, aperture, area and density,leaf area, leaf water potential, proline content and MDA content ofP. euphraticahave significant differ‐ences under different degrees of drought stress. The relationship between soil water content and stomatal aperture, stomatal area, leaf water potential, proline content was found to be closely coupled under severe drought stress conditions. This indicates that stomatal aperture, stomatal area, leaf water potential and pro‐line content were more sensitive to severe water defi‐cit, and thus they can respond quite rapidly to a de‐fense mechanism of severe drought stress conditions.It is important to note that a weaker relationship be‐tween soil water content and leaf area, MDA content and stomatal density were found under mild drought stress. While it is not absolutely clear, a possible rea‐son for this is that mild drought stress has little influ‐ence on leaf area, MDA content and stomatal density,and thatP. euphraticahas the ability to quickly adapt to mild drought stress.

5 Conclusions

P. euphraticacould possibly reduce water evapo‐ration by shutting down the stoma, leading to an im‐provement in its water use efficiency with better sur‐vival under drought stress conditions. However, sto‐matal morphology regulation is not the only way that relaxes the damage of drought stress forP. euphrati‐ca,P. euphraticacan also relax the damage of drought stress conditions by reducing leaf area, decreasing the leaf water potential and accumulating a larger amount of MDA and proline.

Acknowledgments:

Funding for this study was provided by the National Natural Science Foundation of China "Nighttime tran‐spiration and its eco-hydrological effects in typical desert vegetation" (42001038) and the Major special projects of science and technology in Inner Mongolia Autonomous Region (zdzx2018057) and the project of CAS innovation cross team (JCTD-2019-19). The authors thank anonymous reviewers for their valuable review and constructive comments on this manuscript.