plants Article Phosphorous Supplementation Alleviates Drought-Induced Physio-Biochemical Damages in Calligonum mongolicum Abd Ullah 1,2,3,4 , Akash Tariq 1,2,3,4,5,6, * , Fanjiang Zeng 1,2,3,4, *, Jordi Sardans 5,6 , Corina Graciano 7 , Sami Ullah 8 , Xutian Chai 1,2,3,4 , Zhihao Zhang 1,2,3,4 , Maierdang Keyimu 1,2,3,4 , Muhammad Ahsan Asghar 9 , Hafiz Hassan Javed 10 and Josep Peñuelas 5,6 1 Xinjiang Key Desert Plant Roots Ecology and Vegetation Restoration Laboratory, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830000, China 2 State Key Laboratory of Desert and Oasis Ecology, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830000, China 3 Cele National Station of Observation and Research for Desert-Grassland Ecosystems, Cele 848300, China 4 University of Chinese Academy of Sciences, Beijing 100045, China 5 CSIC, Global Ecology Unit, CREAF-CSIC-UAB, Bellaterra, 08193 Barcelona, Catalonia, Spain 6 CREAF, 08193 Cerdanyola del Vallès, Catalonia, Spain 7 Instituto de Fisiología Vegetal, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de La Plata, Buenos Aires B1406, Argentina 8 Department of Botany, University of Peshawar, Peshawar 25000, Pakistan 9 Department of Biological Resources, Agricultural Institute, Centre for Agricultural Research, ELKH, 2462 Martonvásár, Hungary 10 College of Agronomy, Sichuan Agricultural University, Chengdu 611130, China * Correspondence: akash.malik786@mails.ucas.ac.cn (A.T.); zengfj@ms.xjb.ac.cn (F.Z.); Tel.: +86-155-0448-0471 (A.T.) Citation: Ullah, A.; Tariq, A.; Zeng, F.; Abstract: Calligonum mongolicum is a phreatophyte playing an important role in sand dune fixation, Sardans, J.; Graciano, C.; Ullah, S.; but little is known about its responses to drought and P fertilization. In the present study, we Chai, X.; Zhang, Z.; Keyimu, M.; performed a pot experiment to investigate the effects of P fertilization under drought or well-watered Asghar, M.A.; et al. Phosphorous conditions on multiple morpho-physio-biochemical attributes of C. mongolicum seedlings. Drought Supplementation Alleviates stress leads to a higher production of hydrogen peroxide (H2 O2 ) and malondialdehyde (MDA), Drought-Induced Physio-Biochemical Damages in leading to impaired growth and metabolism. However, C. mongolicum exhibited effective drought Calligonum mongolicum. Plants 2022, tolerance strategies, including a higher accumulation of soluble sugars, starch, soluble protein, proline, 11, 3054. https://doi.org/10.3390/ and significantly higheractivities of peroxidase (POD) and catalase (CAT) enzymes. P fertilization plants11223054 increased the productivity of drought-stressed seedlings by increasing their growth, assimilative shoots relative water content, photosynthetic pigments, osmolytes accumulation, mineral nutrition, Academic Editor: Othmane Merah N assimilation, and reduced lipid peroxidation. Our findings suggest the presence of soil high P Received: 12 September 2022 depletion and C. mongolicum high P requirements during the initial growth stage. Thus, P can be Accepted: 7 November 2022 utilized as a fertilizer to enhance the growth and productivity of Calligonum vegetation and to reduce Published: 11 November 2022 the fragility of the hyper-arid desert of Taklamakan in the context of future climate change. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: hyper-aridity; drought; antioxidant mechanism; N metabolism; P fertilization; xerophytes published maps and institutional affil- iations. 1. Introduction The drylands have enormous socioeconomic and scientific values. For generations, Copyright: © 2022 by the authors. arid regions have provided livelihoods to dependent communities through their biological Licensee MDPI, Basel, Switzerland. and cultural diversity [1]. Despite this, global awareness of the importance of arid regions, This article is an open access article distributed under the terms and and the effort required to manage and protect them, remains low [2,3]. Arid lands suf- conditions of the Creative Commons fer irreparable damage due to biodiversity loss, unsustainable use, rigorous agricultural Attribution (CC BY) license (https:// expansion, and global climate change [4]. As a result of such degradation, ground-water creativecommons.org/licenses/by/ resources, valuable plant species, ecosystem productivity, and communities dependent on 4.0/). these systems may be severely affected. Arid lands host ecosystems that are particularly Plants 2022, 11, 3054. https://doi.org/10.3390/plants11223054 https://www.mdpi.com/journal/plants Plants 2022, 11, 3054 2 of 21 susceptible to climate change and unsustainable human activities [5]; hence, understanding their importance, threats to their biodiversity and productivity, and sustainable man- agement is crucial to meeting global biodiversity conservation targets. Many arid and semi-arid regions of the world have been adversely affected by desertification [6]. Soil water and nutrients are closely related to each other, affecting plants’ development, growth, and productivity. These two groundwater components are essential for establishing and maintaining xerophytes [7]. Xerophytes refer to plant species that can survive prolonged periods of drought. They must extend their roots rapidly to acquire groundwater resources and reduce the threat of water deficit and nutrient limitation to adapt to hyper-arid desert conditions. However, juvenile seedlings are particularly vulnerable to water deficit con- ditions [8]. Generally, hyper-arid ecosystems are characterized by shallow water and extreme nutrient limitations (N & P), making spontaneous seedling establishment difficult for xerophytes. They, therefore, threaten natural vegetation renewal. Therefore, it is critical to investigate the response of young xerophytes to water and nutrient availability before their roots acquire groundwater resources. Water and nutrient availability can affect plant morphological and physio-biochemical metabolism. For instance, a lack of soil water and nutrients diminishes the process of photosynthesis via stomatal closure or metabolic changes. This leads to more excessive light energy absorption than the utilization of the photosynthetic apparatus, causing photoinhibition and photo-oxidative stress due to the excessive accumulation of reactive oxygen species (ROS), including hydrogen peroxide (H2 O2 ) and superoxide anion (O2 − ) [9]. In response, plants have developed various protective mechanisms to counterbal- ance excess ROS production and prevent damage to photosynthetic machinery. These mechanisms involve alterations in light-harvesting antennae, the activation of antioxi- dant enzymatic activities (peroxidase [POD], catalase [CAT], superoxide dismutase [SOD], glutathione reductase [GR], etc.), and antioxidant metabolites (carotenoids, glutathione, ascorbate) [9]. Plants can accumulate compatible solutes (i.e., soluble sugars, proline) under drought stress to maintain their cell turgor, gas exchange capabilities, and growth [10]. Plant cells require large amounts of nitrogen (N), a key component of several biomolecules, including proteins, amino acids, nucleic acids, and other important metabolites. N is primarily absorbed by roots in the form of nitrate (NO3 − ), which is transported to leaves for assimilation [11]. The enzyme nitrate reductase (NR) in the cytoplasm reduces NO3 − to NO2 − , which is then transported to chloroplasts for assimilation into NH4 + by the action of nitrite reductase (NiR) [12]. The NH4 + assimilation into glutamine and subsequently into glutamate is carried out by glutamine synthetase (GS) and glutamine oxoglutarate aminotransferase (GOGAT) [12]. Long-term water stress severely inhibits soil N mobility, resulting in a low N uptake, negatively impacting plant growth [13]. In addition, drought stress inhibits the ability of trees to metabolize N by disrupting its metabolizing enzyme activities. The scenario mentioned above indicates that N availability is strongly related to drought since drought severely changes accessibility and mineralization. Plant growth requires P, which accounts for approximately 0.2% of the plant’s dry weight. However, approximately 80% of the total soil-P is immobile and inaccessible [14], inhibiting growth and metabolism. Therefore, the availability and uptake of P are critical to plant metabolism. For instance, in P-depleted plants, photosynthesis is hampered by the lack of P-containing metabolites involved in the primary metabolism [15]. In addition, P nutrition is closely related to NO3 − uptake. For instance, a low P supply may reduce the NO3 − uptake, causing reductions in the plants’ growth and dry matter production. Many studies suggest that P fertilization improves plant growth and productivity under drought stress [9,16–19]. As a result of P supplementation, soil P deficiency is reduced, plants can tolerate stress better, and physiological, morphological, and biochemical processes are altered, leading to increased plant growth [17–20]. The Taklamakan is a hyper-arid desert characterized by low soil organic matter, a high pH, and reduced water content. These factors commonly result in extremely low soil-P availability. It is dominated by perennial xerophytes, which play critical roles in Plants 2022, 11, 3054 3 of 21 stabilizing the ecosystems in both oases and deserts [1]. In recent decades, soil-P depletions, or the severe P nutritional conditions of xerophytes, have frequently been noticed in desert ecosystems [21,22]. In this hyper-arid region, severely P-depleted soils are detrimental to desert vegetation growth and survival [1]. Based on the increased occurrences of future severe drought events and N-deposition, xerophytes are more likely to be affected by soil-P shortages than other ecosystem plants [21]. Still, little is known about how xerophytes react to soil-P availability. Calligonum mongolicum is a perennial shrub belonging to the family Polygonaceae. It is a popular choice for afforestation to fix and stabilize dunes. It is primarily found in the desert-oasis ecotone of the Taklamakan desert in northwestern China. Its assimilative shoots provide highly nutritious food for camels and sheep [23]. This species has been destroyed by overgrazing by local animals and excessive anthropogenic activities, which left the desert ecosystem vulnerable to drifting dunes. Consequently, the revegetation and restoration of Calligonum vegetation are urgently needed to protect the ecosystems of these oases against future climate change. Previously, C. mongolicum has been investigated for its root ecology [24], physiological responses to abiotic factors, including drought under N fertilization [25], and salinity under K supplementation [23]. To the best of our knowledge, no research has been published on whether P application can improve the drought tolerance of C. mongolicum. In arid regions, the seedling planta- tion has been widely used to restore vegetation, since natural restoration is difficult [26]. However, extreme environmental conditions in the hyperarid desert can severely affect the growth and metabolism of plants, particularly at the young seedling growth stages, causing a severe threat to their survival and establishment. This scenario makes planting young seedlings for vegetation restoration challenging in the hyperarid and saline and nutrients deficient (i.e., P) desert environment [1]. Therefore, investigating the impact of P supplementation on drought adaption strategies in young C. mongolicum seedlings will provide a theoretical framework for restoring Calligonum communities and protecting the fragile ecosystem of the Taklimakan desert. Therefore, it is imperative to investigate the impact of P application and drought on the growth of C. mongolicum seedlings to establish conservation strategies to mitigate the negative consequences of future climate change. In this study, we aimed to investigate(a) how P application (+P/−P) affects antioxidant mechanisms, N metabolism, and mineral nutrition of young C. mongolicum under water- stressed (25–30% field capacity) and well-watered (70–75% field capacity) conditions; and (b) the combined factors that influence its morpho-physio-biochemical characteristics in the hyper-arid desert. We evaluated plant growth, photosynthetic pigments, nonstructural carbohydrates and osmolytes, lipid peroxidation and antioxidant enzymes, N metabolism, and mineral nutrients to answer the above questions. 2. Results Our study examined the effects of P fertilization on multiple morpho-physiological and biochemical attributes of C. mongolicum seedlings under well-watered and water-stressed conditions. 2.1. Changes in Growth Characteristics We observed obvious changes in morphological traits under different levels of water and P fertilization (Figure 1A–E). Water-stressed plants displayed significant decreases in assimilative shoots relative water content (RWC; 30.95%), plant height (26.73%), and above- and below-ground biomass (44.29% and 43.31%, respectively), compared to well-watered plants, regardless of P fertilization (Figure 1A–D). In contrast, P fertilization caused a significant increment in assimilative shoots RWC under both water levels, compared to their unfertilized peers (Figure 1A). In water-stressed seedlings, P fertilization significantly increasedbelow-ground biomass (11.68%), as compared to their unfertilized counterparts (Figure 1D). However, P application had no significant impact on plant height and above- above‑ and below‑ground biomass (44.29% and 43.31%, respectively), compared to well‑ watered plants, regardless of P fertilization (Figure 1A–D). In contrast, P fertilization caused a significant increment in assimilative shoots RWC under both water levels, com‑ pared to their unfertilized peers (Figure 1A). In water‑stressed seedlings, P fertilization Plants 2022, 11, 3054 significantly increasedbelow‑ground biomass (11.68%), as compared to their unfertilized 4 of 21 counterparts (Figure 1D). However, P application had no significant impact on plant height and above‑ground biomass under water‑stressed conditions. On the contrary, P fertilization under under ground biomass well‑watered conditions water-stressed significantly conditions. increased On the contrary, plant height P fertilization (7.81%) under andwell-watered above‑ andconditions significantly below‑ground biomassincreased (11.14%plant andheight (7.81%) 17.22%, and above-(Figure respectively) and below- 1B–D). ground biomass (11.14% and 17.22%, respectively) (Figure 1B–D). Figure Figure 1. Changesin (A) assimilative 1. Changesin(A)assimilative shoots shoots RWC,(B)plant RWC, (B) plant height,(C)above‑ground height, (C) above-groundbiomass,biomass, (D) (D) below-ground below‑ground biomass, biomass, and (E)androot‑shoot (E) root-shoot ratioratio inresponse inresponse to to P supplementation,under P supplementation, under well‑wa‑ well- tered and drought‑stressed watered and drought-stressedconditions. conditions.A Adifferent differentletter followingthe letter following themeans means indicates indicates significant significant differences differences(p (p < 0.05) between < 0.05) betweenthe thefour four treatments, treatments, as asdetermined determinedbyby Duncan’s Duncan’s test.test. BarsBars represent represent means means±SD. An An ± SD. overview of of overview thethetwo‑way two-wayANOVA ANOVA assessing theeffect assessing the effect ofof water water (W) (W)andand phosphorus phosphorus (P) (P) treatments treatments isispresented presented in in the right‑handcorner the right-hand cornerof of each each panel. panel. *, ** *and and****** indicate indicate thatthat F values F values were significant at p ≤ 0.05, 0.01, and 0.001, respectively. were significant at p ≤ 0.05, 0.01, and 0.001, respectively. 2.2. Changes in Chlorophyll a and Chlorophyll b Pigments 2.2. Changes in Chlorophyll a and Chlorophyll b Pigments Drought conditions significantly decreased the concentrations of chlorophyll a (Chla) Drought conditions significantly decreased the concentrations of chlorophyll a (Chla) and chlorophyll b (Chlb) (20.87% and 9.10%, respectively) compared to well-watered condi- andtions, chlorophyll b (Chlb) regardless (20.87% and of P fertilization 9.10%, (Figure respectively) 2A,B). However, P compared fertilizationto well‑watered more significantlycon‑ ditions, regardless of P fertilization (Figure 2A,B). However, P fertilization increased the concentrations of Chla (27.07%) and Chlb (12.79%) in water stress morefertil- signifi‑ cantly izedincreased seedlings the thanconcentrations in unfertilizedofseedlings. Chla (27.07%) Chla and and Chlb (12.79%) in Chlb increased bywater 20.21% stress and fer‑ tilized seedlings 25.48% more inthan in unfertilized well-watered seedlings. seedlings ChlaPand treated with Chlb than fertilizer increased those by left 20.21% unfertil-and ized (Figure 25.48% more in2A,B). In addition, well‑watered Chla and seedlings Chlb with treated significantly decreased P fertilizer in water-stressed than those left unfertilized (13.05%) (Figure compared 2A,B). to well-watered In addition, Chla andconditions. However, P fertilization Chlb significantly decreased significantly in- in water‑stressed creased Chla/b ratio (12.74%) in water-stressed seedlings compared to their unfertilized counterparts (Figure 2C). (13.05%) compared to well‑watered conditions. However, P fertilization significan creased Chla/b ratio (12.74%) in water‑stressed seedlings compared to their unfer Plants 2022, 11, 3054 5 of 21 counterparts (Figure 2C). Figure Figure 2.2. Changes Changes ininconcentrations concentrations of chlorophyll of (A) (A) chlorophyll a, (B) chlorophyll a, (B) chlorophyll b and (C) bchlorophyll and (C) chloroph a/b ratio ratio in response in response to to P supplementation, under P supplementation, underwell-watered well‑watered and and drought-stressed conditions. drought‑stressed conditions A different letter following the means indicates significant differences (p < 0.05) between ferent letter following the means indicates significant differences (p < 0.05) between the fou the four treatments, ments, as determinedby as determined by Duncan’s Duncan’s test. Bars test. represent Bars represent ± SD. An meansmeans ±SD.overview of the two- An overview of the tw way ANOVA assessing the effect of water (W) and phosphorus (P) treatments is presented in the ANOVA assessing the effect of water (W) and phosphorus (P) treatments is presented in the right-hand corner of each panel. * and *** indicate that F values were significant at p ≤ 0.05, 0.01, and hand corner of each panel. * and *** indicate that F values were significant at p ≤ 0.05, 0.01, and 0.001, respectively. respectively. 2.3. Biochemical Changes Soluble sugar and starch concentrations exhibited obvious changes in respo different water and P fertilization levels (Figure 3A,B).Drought significantly increase uble sugar (31.31%) but decreased starch (9.90%) compared to well‑watered cond Plants 2022, 11, 3054 6 of 21 2.3. Biochemical Changes Plants 2022, 11, x FOR PEER REVIEW 6 of 23 Soluble sugar and starch concentrations exhibited obvious changes in response to different water and P fertilization levels (Figure 3A,B). Drought significantly increased soluble sugar (31.31%) but decreased starch (9.90%) compared to well-watered conditions, regardless soluble sugarof and P fertilization (Figure 3A,B). starch in well‑watered P fertilization exhibited seedlings:decreased solublethe opposite sugar (7.62%)effect on andin‑ soluble starch creased sugar and starchcompared (11.29%), in well-watered seedlings:decreased with their soluble sugar unfertilized counterparts. (7.62%) However, and- P ferti‑ increased lization starch (11.29%), significantly compared increased solublewith their sugar unfertilized (12.02%), but thecounterparts. However,re‑P starch concentration fertilization significantly increased soluble sugar (12.02%), but the starch concentration mained unchanged in water‑stressed seedlings compared to their unfertilized counter‑ re- mained parts unchanged (Figure 3A,B). in water-stressed seedlings compared to their unfertilized counterparts (Figure 3A,B). Figure 3. Figure Changesinconcentrations 3. Changes inconcentrationsof of(A) (A)soluble solublesugar, sugar, (B) (B) starch, starch, (C) (C) soluble soluble sugar/starch sugar/starchratio, ratio, and (D) nonstructural carbohydrates in response to P supplementation and (D) nonstructural carbohydrates in response to P supplementation under well‑watered and under well-watered and drought-stressed conditions. A different letter following the means indicates significant drought‑stressed conditions. A different letter following the means indicates significant differences differences (p <<0.05) (p 0.05)between betweenthe the four four treatments, as determined by Duncan’s test. Bars represent means ± treatments, as SD. ±SD. An overview of the two‑way ANOVA assessing the effect of water (W) and An overview the two-way ANOVA assessing the effect of water (W) and phosphorus (P) treatmentsphosphorus (P) treat‑ ments is presented is presented in the right‑hand in the right-hand corner ofcorner of each each panel. panel. * and * and *** *** indicate indicate that that F values wereF values were significant at significant at p ≤ 0.05, 0.01, and 0.001, p ≤ 0.05, 0.01, and 0.001, respectively. respectively. The Theratio ratioof ofsoluble solublesugar sugarto tostarch starch (SS/ST) (SS/ST)significantly significantlyincreased increased(45.82%) (45.82%)ininwater‑ water- stressed stressedseedlings seedlingscompared compared to to well‑watered well-watered seedlings, seedlings,regardless of P fertilization regardless (Fig‑ of P fertilization ure 3C).3C). (Figure However, However,SS/ST significantly SS/ST decreased significantly decreased in in well‑watered well-watered(16.99%) (16.99%)butbutincreased increased in in water‑stressed seedlings(9.23%) water-stressed seedlings (9.23%)under underP P fertilization fertilization (Figure (Figure 3C).3C). Nonstructural Nonstructural car‑ carbohy- bohydrates drates (NSCs)(NSCs) werewere moremore significantly significantly increased increased in water‑stressed in water-stressed seedlings seedlings (12.91%) (12.91%) than than in well‑watered in well-watered seedlings. seedlings. NSCsNSCslevelslevels were unaffected were unaffected by P fertilization by P fertilization in well‑ in well-watered seedlings, watered but significantly seedlings, increasedincreased but significantly in water-stressed seedlings (8.67%) in water‑stressed seedlings(Figure 3D). (8.67%) Water- (Figure stressed 3D). significantlysignificantly Water‑stressed decreased the concentration decreased of soluble protein the concentration (51.29%) of soluble but increased protein (51.29%) proline but (36.82%) increased compared proline (36.82%) tocompared the well-watered condition (Figure to the well‑watered 4A).(Figure condition P fertilization in- 4A). P fer‑ creased soluble tilization protein increased (41.66%) soluble proteinin water-stressed seedlings, whereas (41.66%) in water‑stressed it decreased seedlings, whereasproline it de‑ (33.37%)proline creased in well-watered (33.37%) inseedlings (Figure well‑watered 4A,B). (Figure 4A,B). seedlings Plants 2022, 11, x FOR PEER REVIEW 7 of 23 Plants 2022, 11, 3054 7 of 21 Changesinconcentrations Figure4.4.Changes Figure inconcentrationsofof(A) (A)soluble solubleprotein proteinand and(B)(B)proline prolineininresponse responsetotoPPsupple‑ supple- mentation, under well-watered and drought-stressed conditions. A different letter mentation, under well‑watered and drought‑stressed conditions. A different letter following the following the means indicates significant differences (p < 0.05) between the four treatments, as means indicates significant differences (p < 0.05) between the four treatments, as determined by determined by Duncan’s test. Bars represent means ±SD. An overview of the two‑way ANOVA assessing Duncan’s test. Bars represent means ± SD. An overview of the two-way ANOVA assessing the effect the effect of ofwater water(W)(W)and andphosphorus phosphorus(P)(P)treatments treatmentsisis presented presented in in the the right‑hand right-hand corner corner of of each each panel. panel. **, and *** indicate that F values were significant at p ≤ 0.05, 0.01, and 0.001, respectively. ** and *** indicate that F values were significant at p ≤ 0.05, 0.01, and 0.001, respectively. 2.4.Changes 2.4. Changesin inROS ROSAccumulation AccumulationandandLipid LipidPeroxidation Peroxidation Lipidperoxidation Lipid peroxidationwaswassignificantly significantlyhigher higherinin water‑stressed water-stressedthan thanin in well‑watered well-watered Plants 2022, 11, x FOR PEER REVIEW seedlings. The MDA and H O were significantly higher (11.11- and 64.34%, respectively) 8 of 23 seedlings. The MDA and H2O 2 2 2 were significantly higher (11.11‑ and 64.34%, respectively) inwater‑stressed in water-stressed compared compared to well-watered well‑watered seedlings seedlings (Figure (Figure 5A,B). 5A,B).However, However,PPfertiliza- fertili‑ tion significantly reduced the concentration of MDA and H O in well-watered zation significantly reduced the concentration of MDA and H22 22 in well‑watered (35.50% (35.50% and34.78%, and 34.78%,respectively) respectively)andandwater‑stressed water-stressed(48.81‑ (48.81-and and28.45%, 28.45%,respectively) respectively)seedlings, seedlings, comparedto compared totheir theirunfertilized unfertilizedcounterparts counterparts (Figure (Figure 5A,B). 5A,B). Changesininconcentrations Figure5.5.Changes Figure concentrationsofof(A) (A)MDA MDAand and(B) (B) HH22OO2 2ininresponse responsetotoPPsupplementation, supplementation, under well-watered and drought-stressed conditions. A different letter under well‑watered and drought‑stressed conditions. A different letter following the following the means means indicates indi‑ significant differences (p < 0.05) between the four treatments, as determined by Duncan’s cates significant differences (p < 0.05) between the four treatments, as determined by Duncan’s test. test. Bars Bars represent represent means ± SD. means ±SD. AnAn overview overview of the of the two‑way two-way ANOVA ANOVA assessing assessing the the effect effect of water of water (W)(W) and and phosphorus phosphorus (P) treatments (P) treatments is presented is presented in thein right-hand the right‑hand corner corner of each of each panel. panel. * and ** and *** indicate *** indicate that that F values F values werewere significant significant at p at ≤ p0.05, ≤ 0.05, 0.01, 0.01, andand 0.001, 0.001, respectively. respectively. 2.5.Changes 2.5. ChangesininActivities ActivitiesofofAntioxidant AntioxidantEnzymes Enzymes Water stress Water stress significantly significantlyincreased increasedthethe activities of POD activities of (118.61%) and CAT POD (118.61%) and(15.12%), CAT (15.12%), compared to a well‑watered regime, regardless of P fertilization (Figure fertiliza- compared to a well-watered regime, regardless of P fertilization (Figure 6A–C). P 6A–C). Ption had no significant fertilization effects on had no significant SODon effects under SODboth under water bothregimes and POD water regimes andunder water- POD under stressed conditions. P fertilization significantly reduced POD (43.93%) in well-watered water‑stressed conditions. P fertilization significantly reduced POD (43.93%) in well‑wa‑ and CAT tered in well-watered and CAT in well‑wateredand water-stressed seedlings and water‑stressed (25.75% seedlings and 33.89%, (25.75% respectively), and 33.89%, respec‑ compared to unfertilized seedlings (Figure 6B,C). tively), compared to unfertilized seedlings (Figure 6B,C). Plants 2022, 11, x FOR PEER REVIEW 9 o Plants 2022, 11, 3054 8 of 21 Figure 6. Changes in the activity of (A) SOD, (B) POD, and (C) CAT in response to P supplementation, Figure 6. Changes in the activity of (A) SOD, (B)POD, and(C)CAT in response to P supplementa under well-watered and drought-stressed conditions. A different letter following the means indicates under well‑watered and drought‑stressed conditions. A different letter following the means i significant differences (p < 0.05) between the four treatments, as determined by Duncan’s test. Bars cates significant differences (p < 0.05) between the four treatments, as determined by Duncan’s represent means ± SD. An overview of the two-way ANOVA assessing the effect of water (W) and Bars represent means ±SD. An overview of the two‑way ANOVA assessing the effect of water phosphorus (P) treatments is presented in the right-hand corner of each panel. *** indicate that F and phosphorus (P) treatments values were significant is presented at p ≤ 0.05, 0.01, and 0.001,in the right‑hand corner of each panel. * and *** ind respectively. that F values were significant at p ≤ 0.05, 0.01, and 0.001, respectively. 2.6. Changes in Enzymatic Activities of Nitrate Assimilation Water stress significantly decreased − (25.74%) concentration compared to the NO3Assimilation 2.6. Changes in Enzymatic Activities of Nitrate well-watered conditions. In contrast, NH4 + significantly increased under water-stressed Water stress significantly decreased the NO 3− (25.74%) concentration compare conditions, regardless of P fertilization. Further, P fertilization had no significant effect on well‑watered NO3 − and NH4conditions. In contrast, + under well-watered conditions. significantly NH4+Under increased water-stressed under conditions, water‑stres P fertil- conditions, regardless ization significantly enhanced − of P fertilization. the NO3 (22.05%) Further, P fertilization but reduced + the NH4 had no significant (21.56%) concen- effec tration (Figure 7A,B), compared with their unfertilized counterparts. NO3 and NH4 under well‑watered conditions. Under water‑stressed conditions, P f − + Further, waterstress lization significantly enhanced the NO3−(22.05%) but reduced the NH4+ (21.56%) con tration (Figure 7A,B), compared with their unfertilized counterparts. Further, waterst significantly reduced nitrate reductase (NR) activity (66.46%) compared to well‑wate conditions. P fertilization significantly increased NR activity in water‑stressed condit Plants 2022, 11, 3054 9 of 21 022, 11, x FOR PEER REVIEW 10 significantly reduced nitrate reductase (NR) activity (66.46%) compared to well-watered conditions. P fertilization significantly increased NR activity in water-stressed conditions conditions. However, (55.08%). P fertilization Still, it caused significantly no significant increase increasedconditions, under well-watered GS activity in well‑wa compared seedlings withby 32.02%, their unfertilizedcompared with counterparts their (Figure unfertilized 7C). counterparts Water stress had (Figure no significant effect 7D). Fur on GS and GOGAT activities, regardless of P fertilization, as compared to well-watered P fertilization had no significant effect on GS activity under water‑stressed and GO conditions. However, P fertilization significantly increased GS activity in well-watered seedlings by activity32.02%, under both water levels, compared with their unfertilized counterparts (Fi compared with their unfertilized counterparts (Figure 7D). Further, P fertilization 7E). had no significant effect on GS activity under water-stressed and GOGAT activity under both water levels, compared with their unfertilized counterparts (Figure 7E). Figure 7. Changes in concentrations of (A) NO − and (B) NH + , and enzymatic activity of (C) NR 3 4 Figure 7. Changes in concentrations of (A) NO 3− and (B) NH4+, and enzymatic activity of (C (D) GS, and (E) GOGAT in response to P supplementation, under well-watered and drought-stressed (D)GS, and (E)GOGAT in response to P supplementation, under well‑watered and drought‑str conditions. A different letter following the means indicates significant differences (p < 0.05) between conditions. A different the four treatments, letter following as determined the means by Duncan’s indicates test. Bars significant represent means ± SD.differences An overview (p < 0.05) bet of the the fourtwo-way treatments, as determined by Duncan’s test. Bars represent means ±SD. ANOVA assessing the effect of water (W) and phosphorus (P) treatments is presented in theAn overview two‑way ANOVA right-hand assessing corner the effect of each panel. of***water *, ** and (W) indicate thatand phosphorus F values (P) treatments were significant is present at p ≤ 0.05, 0.01, the right‑hand corner of each panel. * and *** indicate that F values were significant at p ≤ 0.05 and 0.001, respectively. and 0.001, respectively. 2.7. Changes in Mineral Nutrients Waterstress significantly altered the concentration of mineral nutrients. N, P, K Plants 2022, 11, 3054 10 of 21 2.7. Changes in Mineral Nutrients Waterstress significantly altered the concentration of mineral nutrients. N, P, K, and Mg concentrations significantly reduced (8.30-, 12.87-, 48.71-, and 47.53%, respectively) in water- stressed seedlings, compared to well-watered, regardless of P fertilization (Figure 8A–D). In both well-watered and water-stressed conditions, P fertilization significantly increased the concentrations of N (4.225% and 5%, respectively) and P (41.77% and 21.53%, respectively), Plants 2022, 11, x FOR PEER REVIEW compared to their unfertilized counterparts (Figure 8A,B). Contrarily, P fertilization11 of not did 23 cause significant changes in K and Mg concentrations, compared with their unfertilized counterparts, regardless of water availability (Figure 8C,D). Changes Figure8.8.Changes Figure in in thethe concentrations concentrations of (A) of (A) TN,TN, (B) (C) (B) TP, TP, K, (C)and K, and (D) Mg(D)inMg in response response to P to P sup‑ supplementation, under well-watered and drought-stressed conditions. A different plementation, under well‑watered and drought‑stressed conditions. A different letter following the letter following the means means indicates indicates significant significant differences differences (p <(p0.05) < 0.05) between between thethe four four treatments,asasdetermined treatments, determinedby by Duncan’stest. Duncan’s test.Bars Barsrepresent representmeans ± SD. means±SD. AnAn overviewofofthe overview thetwo‑way two-wayANOVA ANOVAassessing assessingthetheeffect effect ofofwater water(W) (W) and and phosphorus phosphorus (P) treatments treatments is is presented presentedin inthe theright-hand right‑handcorner cornerofofeach eachpanel. panel.*, *** and and*** ***indicate indicatethat thatFFvalues valueswere weresignificant significantat atpp≤≤0.05, 0.05,0.01, 0.01,and and0.001, 0.001,respectively. respectively. 2.8.Relationship 2.8. Relationshipbetween betweenthe theStudied StudiedParameters Parameters Thecorrelation The correlationanalysis analysisrevealed revealedaastrongnegative strongnegativecorrelation correlationof of proline, proline, HH22OO2,2 ,SS, SS, SS/ST, NSCs, POD, and NH + with+assimilative shoots RWC, ABGB (above-groundbiomass), SS/ST, NSCs, POD, and4 NH4 with assimilative shoots RWC, ABGB (above‑ BGB (below-groundbiomass), groundbiomass), PH (plantheight),PH BGB (below‑groundbiomass), Chl-a and Chl-b, Chl‑a (plantheight), and SP (soluble and Chl‑b, protein) and SP (Figure 9). (soluble The NR, protein) GS, and (Figure 9).GOGAT The NR,exhibited GS, anda strongpositive correlation GOGAT exhibited with assimila- a strongpositive tive shootswith correlation RWC, ABGB, BGB, assimilative PH, Chl-a shoots RWC,and Chl-b, ABGB, SP, and BGB, PH, ST (starch). Chl‑a At theSP, and Chl‑b, same andtime,ST theirnegative correlation was observedwithproline, SS (solublesugar), SS/ST, (starch). At the same time, theirnegative correlation was observedwithproline, NSCs, MDA, SS H2 O2 , SOD, POD, (solublesugar), CAT, SS/ST, NSCs, NH4 + (Figure and MDA, 9). POD, CAT, and NH4+ (Figure 9). H2O2, SOD, nts 2022, 11, x FOR PEER REVIEW 12 of 23 Plants 2022, 11, 3054 11 of 21 FigureFigure 9. Pearson correlation 9. Pearson correlation analysis analysisbetween studyparameters. between study parameters. Assimilative Assimilative shootsshoots RWC (relative RWC (relative water water content), ABGB content), ABGB (above‑ground (above-groundbiomass), BGB(below-ground biomass), BGB (below‑ground biomass), biomass), PH (plant PH (plant height),height), RSR (root‑shoot ratio), RSR (root-shoot Chl−a ratio), Chl−(chlorophyll a),Chl a (chlorophyll a), Chl−b chlorophyll −b chlorophyll b, Chla/b b, Chla/b (chlorophyll (chlorophyll a/bSP a/b ratio), ratio), SP (soluble protein), (soluble proline protein), proline SSSS(soluble (soluble sugar), ST(starch), sugar), ST (starch), SS/ST SS/ST (soluble (soluble sugar/starch sugar/starch ratio), NSCs ratio), NSCs (nonstructural carbohydrates), (nonstructural carbohydrates), TPTP(Total (Total P), TN TN(total (totalN),N), K (potassium), K (potassium), Mg (magnesium), Mg (magnesium), MDA MDA (malondialdehyde), (malondialdehyde), H2O H22,O(hydrogen 2 , (hydrogenperoxide), peroxide), SOD SOD(superoxide (superoxide dismutase),CAT dismutase),CAT (catalase), POD POD (catalase), (peroxidase), NO − (nitrate), NH + (ammonium), NR (nitrate reductase), GS (glutamine synthetase), (peroxidase), NO3 (nitrate), − 3 NH4 (ammonium), + 4 NR (nitrate reductase), GS (glutamine synthetase), GOGAT GOGAT (glutamine (glutamine oxoglutarate aminotransferase). * p* ≤ oxoglutarateaminotransferase). p ≤0.05, ** p**≤p0.01, 0.05, ≤ 0.01, ≤ 0.001. *** p*** p ≤ 0.001. 2.9. Results Summary 2.9. Results WeSummary observed a positive interaction between water and P supply on the most studied We observed variables a positive related interaction to plant fitness betweenbiomass, (below-ground water and P supply height, on the chlorophyll, NH4 +studied andmost content) variables and storing related to plant(starch) resources fitness (Table S1).biomass, (below‑ground In contrast, we observed height, a negative chlorophyll, and NH4+ interaction between water and P supply in the most studied variables related to stress content) and storing (starch) resources (Table S1). In contrast, we observed a negative avoidance (CAT, POD, soluble sugars, proline) and H2 O2 production (Table S1). Thus, the interaction between interaction analyseswater and strongly P supply suggest that a in the most positive studied synergy enhancesvariables relatedand growth capacity to stress avoidance (CAT, resource POD, soluble production when watersugars, proline) and and phosphorus areHenhanced. 2O2 production (Table Contrarily, S1).both when Thus, the interaction analyses strongly suggest that a positive synergy enhances growth capacity resources are supplied together, there exists a synergy decreasing plant stress. and resource production when water and phosphorus are enhanced. Contrarily, when 3. Discussion both resources are supplied together, there exists a synergy decreasing plant stress. Our findings demonstrated that C. mongolicum was adversely affected by drought in terms of reduced assimilative shoots RWC, shoot and root biomass, chlorophyll pigments, 3. Discussion mineral nutrition, N metabolism, and ROS and MDA concentrations. However, P fertiliza- tion contributed Our positively to thethat findings demonstrated growth and metabolism C. mongolicum of C. was mongolicum, adversely which indicates affected by drought in that it requires high levels of P during the seeding stage of its growth. terms of reduced assimilative shoots RWC, shoot and root biomass, chlorophyll pigments, mineral nutrition, N metabolism, and ROS and MDA concentrations. However, P fertilization contributed positively to the growth and metabolism of C. mongolicum, which indicates that it requires high levels of P during the seeding stage of its growth. Plants 2022, 11, 3054 12 of 21 3.1. P Fertilization Improvedthe Growth and Biomass of Young Seedlings Our study found that plant height and above- and below-ground biomass was signifi- cantly decreased in water-stressed compared to well-watered seedlings, suggesting that C. mongolicum is highly sensitive to soil water shortages. When the soil available-P concen- tration cannot meet the demands of plant growth, plants can respond to the P deficiency by reducing biomass accumulation [27]. Our findings are consistent with previous studies on different plant species [9,20,28,29]. However, P fertilization resulted in a significant increase in the height and biomass under well-watered conditions, suggesting that C. mongolicum has high P demands and that the soil in the hyper-arid desert lacks enough P to meet the plant’s functional re- quirements [30]. These findings are consistent with previous studies reporting increased growth rates and biomass accumulation in plants fertilized with P under moist soil con- ditions [9,20,31]. However, P application had no significant impact on plant height and above-ground biomass under water-stressed conditions. In contrast, P fertilization sig- nificantly increased assimilative shoots RWC and below-ground biomass; however, the percentage increase was lower than the effect of P fertilization under well-watered con- ditions (9.86 < 12.19% and 11.68 < 17.22%, respectively). This scenario indicates that P mobility strongly decreases in water deficit conditions [32]. Previous studies exhibited that fertilization on plant growth gradually decreases with decreases in soil moisture and even disappears completely under extremely dry conditions [33]. Further, we observed that P fertilization resulted in an increased root-shoot ratio of water-stressed seedlings compared to well-watered seedlings. Higher root biomass con- tributes to drought tolerance by increasing the roots’ ability to extract soil moisture [34,35]. We suggest that P fertilization enhances the growth and root biomass of young water- stressed C. mongolicum, thereby increasing the shrub’s drought tolerance in a hyper-arid, P-deficient soil condition. In hyperarid desert conditions, xerophytes require a large root system to quickly exploit groundwater resources, reduce the risk of water and nutrient deficiencies, and ensure their survival [36]. 3.2. Improved Assimilative Shoots Relative Water Content (RWC) The relative water content (RWC) of leaves reflects the balance between the water supply to the leaves and the transpiration rate. Therefore, it is considered an important indi- cation of the water status of plants [37]. In our study, assimilative shoots RWC significantly decreased under water-stressedconditions compared with the well-watered condition. A reduction in leaf RWC under water-stressed conditions has also been observed in various plant species [9,17,18,38], including C. mongolicum [25]. P addition has been shown to increase plants’ leaf relative water content (LRWC) in water-stressed plants [39,40]. Like- wise, our study exhibits that P fertilization significantly increased the assimilative shoots RWC of C. mongolicum, regardless of water availability. We speculate that the P application may have allowed C. mongolicum to accumulate more osmolytes, compared to unfertilized plants [9]. The higher concentrations of osmolytes could have altered the osmotic potential and facilitated water movement in the assimilative shoots. In previous studies, increased plant RWC has been attributed to either the improvement in the root’s ability to absorb water or nutrients or an improved ability of the plant’s tissues to conserve water [17,40]. 3.3. P Fertilization Improved Photosynthetic Chlorophyll Pigments In our study, photosynthetic pigment concentrations (Chla and Chlb) were significantly lower in water-stressed than well-watered seedlings, regardless of the P application. This scenario indicates that oxidative stress damages photosynthetic pigments in water-stressed seedlings. Previous studies demonstrated that Chla and Chlb are sensitive to soil water shortage [9,17,31,38] and reported that drought stress could damage photosynthetic pig- ments. A previous study [41] demonstrated that soil dehydration damages the lamellae vesiculation and chloroplast membranes, which results in decreased chlorophyll concentra- tion. Therefore, we suggest that a water deficit condition had caused the degradation of Plants 2022, 11, 3054 13 of 21 photosynthetic pigments, leading to the diminished photosynthetic capacity and growth of C. mongolicum seedlings. P fertilization significantly increased the concentrations of chlorophyll pigments in C. mongolicum under both water regimes. Previous studies reported that P fertilization increases photosynthetic pigment synthesis in plants during drought, corroborating our findings [9,38]. In water-stressed plants, improved photosynthetic pigments have been shown to increase light harvestingefficiency, thus resulting in a higher net photosynthetic rate (Pn) [9]. In contrast, someother P fertilization studies reported a negligibleimprove- mentin chlorophyll concentrations of water-stressed plants due to variations in experimen- tal duration, levels and durations of water deficits, and the drought tolerance ability of a particular plant species [42,43]. Although, our results are consistent with previous studies, which also suggest that the application of P improves photosynthetic pigment synthesis in water-stressed plants [19,20,38]. 3.4. P Fertilization Increased Soluble Protein, and Carbohydrate Concentrations We found that the soluble protein concentration was lower in water-stressed than in well-watered seedlings. Some potential explanations for this result include an increase in protease enzyme activity, increased proteolysis, or reduced protein synthesis, along with a lower photosynthetic rate (i.e., less carbon to build metabolites), in water-stressed plants. Water stress significantly increased the proline concentration compared to well-watered conditions. A high proline accumulation may reflect osmotic adjustments under drought stress and aid in ROS detoxification, contributing to membrane and macromolecule protec- tion [44]. It has been reported that proline accumulates under low moisture conditions due to a reciprocal mechanism involving the activation of enzymes required for its synthesis and the suppression of enzymes required for its degradation [45]. Nonstructural carbohydrates (NSCs), such as soluble sugars and starches, are the products of plant photosynthesis. NSCs indicate the amount of substance needed for plant survival, growth, and buffering capacity under adverse environmental conditions [46] and therefore play a critical role in regulating plant physiological phenomena in response to stress conditions. In our study, NSCs concentrations in assimilative shoots varied significantly in response to water and P fertilization, which could be attributed to the physiological mechanisms and the changes in the NSC components. We observed a lower starch concentration and higher concentrations of soluble sugar, NSCs, and soluble sugar/starch ratios in water-stressed seedlings than in well-watered seedlings, irrespective of P treatment. Drought stress causes stomatal closure and retrains photosynthetic C gain, forcing the plants to use starch reserves for metabolic requirements [8,47]. Likewise, our study demonstrated that water-stressed C. mongolicum maintained a high ratio of soluble sugar to starch in assimilative shoots, regardless of P application. Therefore, water-induced increases in soluble sugar and reductions in starch within the assimilation shoots of C. mongolicum may be explained by converting stored starch into soluble sugars [48] for osmotic purposes adjustment. These results are not surprising, since plants in hyper-arid desert conditions (water deficit) require more soluble sugars for metabolism, since water deficit inhibits the important process of photosynthesis and reduces CO2 assimilation but does not substantially reduce respiration rates [49]. A sufficient concentration of soluble sugars is essential for osmotic adjustment for optimal phloem turgor to maintain phloem transport and refill xylem embolism when there is a water shortage [50]. P fertilization has been reported to play a critical role in regulating carbohydrate con- centrations under drought stress conditions by altering the activity of correlative enzymes in carbohydrate metabolism [18,51]. In our study, soluble sugar levels were significantly higher in P-fertilized seedlings than in non-fertilized seedlings, which is consistent with previous findings [9,38]. Plants 2022, 11, 3054 14 of 21 3.5. P Fertilization Reduced Lipid Peroxidation, ROS Production, and Antioxidant System Drought stress often induces the formation of ROS, including anion radicals (O2 − ) and hydrogen peroxide (H2 O2 ), which react with lipids, proteins, and DNA and lead to oxidative damage and the disrupting of normal cell function and a serious reduction in plant growth [52,53]. In our study, there was a significant increase in H2 O2 concentrations in water-stressed seedlings, regardless of P fertilization, which may reflect a decrease in photosynthetic rate and other physiological changes occurring due to drought stress, seemingly corroborated by correlation analysis (Figure 9). The elevated ROS production resulted in significant membrane lipid peroxidation damage, as demonstrated by the significantly increased MDA concentration in the water-stressed C. mongolicum seedlings compared to the well-watered seedlings. Excessive ROS also damage protein structures and inactivate enzymes that function in various physiological processes [54]. Plant antioxidant enzymes, such as SOD, POD, and CAT, are essential in maintaining a dynamic equilibrium by scavenging excess ROS. The antioxidant enzyme activity of plants can be correlated directly with the ability of the plant to scavenge ROS (Figure 9). Our study showed that water-stressed seedlings had significantly higher enzyme activities (SOD, POD, and CAT) than well-watered seedlings, regardless of P fertilization. Similar results were also reported in other studies conducted on various plant species [9,55]. P fertilization did not change SOD activity under both water regimes and significantly decreased POD activity under water-stressed conditions, whereas CAT was significantly reduced in both water regimes, compared to non-fertilized seedlings. We suggest that C. mongolicum significantly increase POD and CAT activities to scavenge ROS in response to water stress. In contrast, applying P helps to prevent ROS-induced damage, as demon- strated by reduced MDA and improvements in other physiological functions. 3.6. P Fertilization Increased N-Metabolism by Increasing Its Assimilative Enzymes Plants readily absorb and utilize two forms of soil N (NO3 − and NH4 + ) in their growth. Drought stress has been associated with changes in the metabolism of N by altering the activity of N metabolic enzymes in previous studies [56,57]. Changing enzyme activity can significantly affect the efficiency of N uptake and utilization. In our study, water stress significantly declined NO3 − concentration, compared to well-watered conditions. NO3 − promotes plant growth, osmotic regulation, and assists plants in adapting to abiotic stress conditions [58]. It is also crucial for stomatal closure by affecting the depolarization of guard cells [59]. Nitrate reductase (NR) activity was more significantly reduced in water-stressed conditionsthan in well-watered conditions. The reduction in NR might be attributed to a reduction in leaf photosynthesis, resulting from stomatal closure, which inhibits growth. A previous study [60] reported significant reductions in the activity of NR and NiR enzymes and the expression levels of GmNR and GmNiR in soybean leaves leading to impaired nitrogen assimilation. Several studies have claimed that drought stress decreases NR activity [61–63]. Decreasing NR activity may have some advantages for plants since it contributes rapidly to maintaining osmotic pressure in photosynthetic cells through an increase in the NO3 − concentration. NH4 + is assimilated by glutamine synthase (GS) and glutamine oxoglutarate amino- transferase (GOGAT). The GS/GOGAT metabolism cycle is inhibited by drought stress, which weakens NH4 + assimilation, causing cell death and toxicity [58,60]. In our study, NH4 + significantly increased under water-stressed, compared to well-watered conditions, regardless of P fertilization. Further, water stress did not significantly affect GS/GOGAT metabolism in our study (Figure 7D,E), indicating that C. mongolicum seedlings accelerated NH4 + to withstand drought stress. A recent study demonstrated that GS activity remained unchanged in S. japonica seedlings under drought stress, while GOGAT activity increased in both roots and leaves [62]. Furthermore, previous studies have demonstrated that P application can adaptively regulate N metabolism under water stress by modulating the activity of its associated enzymes [18,57,63]. We observed that P fertilization increased the Plants 2022, 11, 3054 15 of 21 NR activity under drought stress, whereas there was no significant effect on NR under well-watered and GS/GOGAT activities under both stress conditions, compared with their unfertilized counterparts. NH4 + is not only a vital component of living cells, but also an essential intermediate in plant metabolism. However, it can adversely affect the metabolism of higher plants when present in excess [64]. In our study P fertilization significantly reduced NH4 + in water- stressed conditions (Figure 7B), compared with their unfertilized counterparts. Therefore, we suggest that P fertilization might have decreased or prevented toxic effects associated with the excessive NH4 + accumulation under water stress [18]. A previous study [18] reported that glutamate dehydrogenase (GDH) activity under P-fertilized water-stressed seedlings was higher than in well-watered conditions. We propose that P fertilization under waterstress might have increased the GDH activity, which had accompanied the GS/GOGAT cycle within C. mongolicum, for detoxifying excess NH4 + . 3.7. P Fertilization Improved Mineral Nutrients We observed significant reductions in the concentration of assimilating shoot N, P, K, and Mg in water-stressed seedlings, compared to well-watered. Water availability plays a key role in nutrient uptake and assimilation, and several studies demonstrated that decreased soil moisture reduces mineral nutrient uptake and concentrations [28,65], which corroborates our findings. Compared to their unfertilized counterparts, P fertilization resulted in significant increases in the assimilation of shoot N and P, while it did not significantly affect the concentrations of K and Mg. Previous studies reported that P fertilization increases plant N and P concentrations [66,67]. Both N and P contribute to the process of photosynthesis, cell growth and metabolism, and protein synthesis [68]. Hence, we suggest that a P-fertilization-induced increase in shoot N and P concentrations might have improved photosynthesis and protein synthesis, resulting in the improved growth of C. mongolicum, compared with their unfertilized counterparts. 4. Materials and Methods 4.1. Experiment Design The present study was conducted between June and early October 2021 in an outdoor nursery at the Cele National Station of Observation and Research for Desert-Grassland Ecosystem (37◦ 000 56” N, 80◦ 430 81” E), Chinese Academy of Sciences, located at the southern edge of the Taklamakan Desert, Northwest China. This region is extremely hyper-arid, with a mean yearly temperature and precipitation of 11.9 ◦ C and 35 mm, respectively. At least eight seeds collected from the natural desert were sown in each 25 L pot (ca. 18%), filled with 20 kg of homogenized soil (aeolian loamy sand with organic C, 2.99 g kg−1 ; total N, 0.23 g kg−1 ; total P, 0.60 g kg−1 and total K, 23.11 g kg−1 ). The pots were arranged in a complete randomized design (RCD) and watered regularly. 4.2. Treatments Application Following the seedlings’ establishment under regular management for one month, they were thinned to one healthy and uniform seedling per pot and exposed to four treatments for three months: two levels of water treatments (well-watered [control 70–75% field capacity] and water-stressed [severe drought 25–30% field capacity]) and two rates of P fertilization (+P and −P). There were 12 replications of each treatment. We calculated the soil relative water content (SRWC) for the two water treatments using a weighted method [9]. We applied sodium di-hydrogen phosphate (NaH2 PO4 , 25.5% P) as a P fertilizer, with the dose consisting of 390 mg P mixed in 400 mL of water per pot. Five rounds of fertilization were carried outafter every 20 days. Besides, Pots were rotated once a week throughout the experiment to avoid the possibility of systematic errors associated with variations in environmental conditions. At the end of the experiment, all of the plants were harvested. Shoot samples are immediately put in Ziploc bags and stored at −80 ◦ C for physio-biochemical determination in the laboratory. Plants 2022, 11, 3054 16 of 21 4.3. Growth Parameters 4.3.1. Determination of Assimilative Shoot Relative Water Content A single sample of assimilative shoots from each plant was collected, and their fresh weight (FW) was determined. We immediately dipped the samples into distilled water at a temperature of 4 ◦ C for 4 h in the dark to measure the turgid weight (TW). To determine the dry weight (DW), the samples were placed in a 75 ◦ C oven for 24 h. The assimilative shoot relative water content (LRWC) was calculated by using the following equation: Assimilative shoot RWC = [(FW − DW)/(TW − DW)] × 100% 4.3.2. Measurement of Plant Growth and Biomass The plant height (cm) was measured using a measuring tape. The above- and below- ground plant parts were separated and weighed after being removed from the soil. We calculated R/S (root-shoot) ratios by dividing the biomass of the plants’ above- and below- ground parts by the ratio of their dry weights. 4.4. Physio-Biochemical Analysis 4.4.1. Measurement of Photosynthetic Pigments Fresh assimilating shoots were immersed in 95% ethanol (v/v) to extract the photo- synthetic chlorophyll pigments. We determined the amount of chlorophyll a (Chla) and chlorophyll b (Chlb) by reading the absorbance at 665 nm and 649 nm, respectively [13], using a spectrophotometer (Type UV-754; Shanghai Accurate Scientific Instrument Co., Shanghai, China). The chlorophyll concentrations were determined using the following equations (mg g−1 FM). Chla = 13.98 A665 − 6.88 A649 (1) Chlb = 24.96 A649 − 7.32 A665 (2) Chla/b = Chl a/Chlb (3) Chl = Chla + Chlb (4) 4.4.2. Determination of Nonstructural Carbohydrates Dried shoot samples were ground into a fine powder using a ball mill. The colorimetric determination of the soluble sugar concentration was determined using glucose, following a standard method [69]. Starch was measured calorimetrically at 620 nm, with a slight modification in the anthrone method [70]. The NSC represented the sum of soluble sugars and starch contents. 4.4.3. Determination of Soluble Protein and Proline The proline extraction (0.2 g fresh assimilative shoot) was conducted using two mL of 10% acetic acid and five mL of 3% salicylic acid, respectively. The resulting supernatants were analyzed according to a standard method [61]. Approximately 0.3 g of fresh assim- ilative shoots were used to determine the concentration of soluble proteins using bovine serum albumin as the standard [71]. 4.4.4. Oxidative Stress Indicators Hydrogen peroxide (H2 O2 ) concentrations were determined by a standard proce- dure [72]. The 0.2 g freshly assimilative shoots were homogenized in 5 mL of trichloroacetic acid (0.1%) in an ice bath, transferred to tubes, and centrifuged at 5000× g for 10 min (4 ◦ C). Next, we centrifuged the supernatant comprising of 0.1 mL of titanium reagent (50 µL of 20% titanium tetrachloride) and 0.2 mL of ammonia, centrifuged at 10,000× g for 10 min. Following five washes with acetone, the precipitate was centrifuged at 10,000× g for 10 min, after which 3 mL of 1 M H2 SO4 was added. The absorbance was read at 410 nm using a spectrophotometer (Type UV-754; Shanghai Accurate Scientific Instrument Co., Shanghai, China). Plants 2022, 11, 3054 17 of 21 The malondialdehyde (MDA) concentration was assessed based on the thiobarbituric acid (TBA) test for the evaluation of lipid peroxidation [73]. Fresh assimilative shoot samples (0.5 g) were homogenized in 1 mL of 5% trichloroacetic acid (TCA) and centrifuged for 10 min at 5000× g (4◦ C). Using a separate tube, 4 mL of the supernatant was added to 2 mL of 20% TCA (containing 0.5% TBA), and the mixture was heated at 100 ◦ C for 15 min before centrifugation at 5000× g for 10 min. Using a spectrophotometer, OD450, OD532, and OD600 nm were determined, and the concentration of MDA was calculated according to the following equation: MDA (mol g−1 FW) = 6.45 (OD532 − OD600) − 0.56OD450. 4.4.5. Antioxidant Enzyme Activities Approximately 0.5 g of fresh assimilative shoot samples were homogenized in liquid nitrogen and 10 mL of 50 mM sodium phosphate buffer (pH 7.8) containing 0.5 mmol EDTA and 0.15 mol NaCl to determine enzyme activity. After centrifuging the homogenate at 12,000× g at 4 ◦ C (20 min), the supernatant was used to measure the activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). We measured the activity of superoxide dismutase (SOD) following a standard method [74]. A reaction mixture of 3 mL was prepared using 50 mM phosphate buffer (pH 7.8), 75 mM of nitro-blue tetrazolium (NBT), 130 mM of methionine, and 2.0 mM of riboflavin, and 0.1 mL of enzyme extract. We assessed the activity of SOD by reading the absorbance at 560 nm using a spectrophotometer (Type UV-754; Shanghai Accurate Scientific Instrument Co., Shanghai, China). The unit of SOD activity was defined as the amount of enzyme required for the 50% inhibition of nitroblue tetrazolium (NBT) reduction. Peroxidase activity (POD) was determined using a standard method [75] with slight modifications. A reaction mixture of 3 mL was prepared by mixing phosphate buffer (pH 6.0), 3 mmol peroxide L−1 , and 0.25% (v/v) of guaiacol with the enzyme extract. Using a spectrophotometer (Type UV-754; Shanghai Accurate Scientific Instrument Co., Shanghai, China), absorbance (470 nm) was read at 1-min intervals for 3 min. The enzyme activity was expressed as U/g·min−1 . The Catalase (CAT) activity was evaluated following a previous method [76] with minor modifications. In brief, a 3 mL reaction mixture was prepared by mixing 0.1 mL of the enzyme extract with 100 mM phosphate buffer (pH 7.0) and 10 mM hydrogen peroxide. We used a spectrophotometer (Type UV-754; Shanghai Accurate Scientific Instrument Co., Shanghai, China), to measure the change in OD240 at 1-min intervals for 3 min. The calculation of CAT activity was carried out using the 39.4 mM−1 ·cm−1 extinction coefficient. The enzyme activity was expressed as U/g·min−1 . 4.4.6. Determination of N-Metabolizing Enzymes The nitrate reductase (NR) activity was determined by homogenizing 0.2 g using 2 mL of 25 mM phosphate buffer saline (PBS, pH 8.7), which contained 10 mM cysteine and 1 mM EDTA, and then centrifuging for 20 min at 30,000× g. The resulting supernatant was tested for NR activity using a diazo-coupling method employing the Griess reagent [13]. The GS activity was determined by homogenizing them in 2 mL of 50 mM Tris-HCl buffer (pH 7.8; containing 15% glycerol, 0.1% TritonX-100, 1 mM of EDTA, and 14 mM of 2-mercaptoethanol) and centrifuging them twice at 4 ◦ C for 10 min. After complexing with acidified ferric chloride, the supernatant was used to determine GS by forming glutamine hydroxamate using a 540 nm fluorescence measurement [13]. 4.4.7. Determination of Mineral Elements We measured the concentrations of mineral nutrients in finely powdered, assimilative shoot samples. The concentration of N and P was determined following digestion with con- centrated H2 SO4 using a Kjeldahl Nitrogen Analyzer (K1160, Jinan Hanon Instruments Co. Ltd., Jinan, China) and a Thermo-Elemental inductively coupled plasma optical emission Plants 2022, 11, 3054 18 of 21 spectrometer (iCAP 6300). Assimilative shoot samples were soaked in HNO3 and HF–HNO H2 O2 , respectively, and then the concentrations of K and Mg were determined [77]. 4.5. Statistical Analysis Measurements were replicated three times and sorted using Microsoft Excel 2019. SPSS version 16.0 (Chicago, IL, USA) was used to perform descriptive statistics two-way analysis of variance (ANOVA) considering water availability and P application as main factors, with two levels for each. Duncan’s multiple range tests were used to compare means at a significance level of 0.05. GraphPad Prism 8 was used to create the figure graphics. For interpretation purposes, Pearson correlation analyses were conducted using OriginPro 2019 software (OriginLabCarporation Northampton, Northampton, MA, USA) regarding the growth parameters, chlorophyll pigment concentrations in the shoots, N metabolism, osmolytes accumulation, mineral nutrition, and reactive oxygen species production rate, as well as antioxidant enzymatic activities. 5. Conclusions Drought adversely affected the growth and metabolism of C. mongolicum, as evidenced by a reduction in assimilative shoots RWC, shoot-root biomass, photosynthetic pigments, mineral nutrition, N metabolism, and an increase in the concentration of ROS and lipid peroxidation. Even so, the improved root-shoot ratio, osmotic solutes (soluble sugar, pro- line), and upregulation of antioxidant enzymes (CAT and POD) suggest that C. mongolicum drought tolerance strategies for maximizing water absorption and reducing oxidative dam- age are effective. In addition, P fertilization positively affected the growth and metabolism of C. mongolicum, which indicates its high P needs at the young seedling stage of develop- ment. In drought-stressed seedlings, P fertilization increased the seedling’s productivity by increasing assimilative shoots RWC, photosynthetic pigments, osmolytes, mineral nu- trition, and N assimilation. In addition, P fertilization reduced the levels of H2 O2 and MDA, leading to reduced POD and CAT activities under both water levels. P fertilization also positively affected the growth and metabolism under well-watered conditions, which indicated the soil’s high P depletion in the hyper-arid desert. Therefore, we suggest that P fertilization played a vital role in the growth and metabolism under well-watered conditions, thus helping in the maintenance of drought tolerance of C. mongolicum, largely through physiological rather than morphological adap- tations. Under both water regimes, the P fertilization increased the below-ground biomass significantly, which may be helpful to young C. mongolicum plants for acquiring ground- water resources and reduce the risk of water and nutrient deficiency in the hyper-arid Taklamakan desert. The addition of water and P had synergistic positive effects on variables related to fitness and energy storing and negative effects on variables related to stress, strongly suggesting the positive impact of both factors on each other. This promising result can help improve field conditions and the management of C. mongolicum communities in the Taklamakan desert. However, further research is needed to elaborate on the un- derlying molecular mechanism operating under drought stress conditions to enhance the understanding of the drought-tolerance capacity of young C. mongolicum. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants11223054/s1, Table S1: Two-way factorial ANOVA. Author Contributions: Conceptualization, A.U. and F.Z.; data curation, A.U.; writing original draft— A.U.; methodology, A.U. and F.Z.; formal analysis and investigation, A.U., M.A.A., J.P., H.H.J. and M.K.; review & editing, A.T., C.G., J.S., J.P., S.U., M.A.A., Z.Z., M.K. and X.C.; funding acquisition, F.Z. and A.T. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the National Natural Science Foundation of China (No. 41977050, 32250410301), the Key Program of Joint Funds of the National Natural Science Foundation of China, and the Government of Xinjiang Uygur Autonomous Region of China (Nos. U1903102). This research was further supported by the Ministry of Science and Technology, China (QN2022045005). Plants 2022, 11, 3054 19 of 21 Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. 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