1 Introduction

Water and nutrients are essential for plant growth, survival, and distribution. In desert ecosystems, these two components are essential for the establishment and maintenance of phreatophytes (Arndt et al. 2004). For adaptation to arid environments, plants need rapid root elongation to obtain groundwater resources and minimize the risk of water and nutrient deficiencies (Canham et al. 2015). Seedlings, however, are more vulnerable to water deficits (McDowell et al. 2008), since their roots are small and cannot access the groundwater. This limits the spontaneous emergence of phreatophyte seedlings and inhibits the regeneration of natural vegetation (Tariq et al. 2022). Therefore, it is crucial to understand how phreatophyte seedlings respond to the availability of water and nutrients before reaching groundwater resources.

Drought conditions lead to a reduction in photosynthesis and carbon fixation, causing disturbances in carbohydrate metabolism and the distribution of dry mass (Chaves et al. 2002). Consequently, water-stressed plants deplete their stored reserves, including starch, due to increased respiration (Sami et al. 2016). This imbalance between the accumulation and utilization of photo-assimilates (Abid et al. 2016), affects the activity of carbohydrate-metabolizing enzymes, with stress triggering the upregulation of starch-degrading enzymes like alpha-amylase (AMY) and beta-amylase (BAM) to enhance soluble sugar production for osmotic balance (Du et al. 2020).

Furthermore, the enzyme soluble acidic (SAI) catalyzes sucrose hydrolysis into fructose and glucose (Li et al. 2002), with its low activity contributing to enhanced sucrose accumulation for osmoregulation in stressed leaves (Kaur et al. 2007). Studies, such as those conducted by Liu et al. (2004) on soybean and Xu et al. (2015a) on rice, demonstrate that drought stress can reduce SAI and sucrose synthase (SuSy) activity, respectively, resulting in increased sucrose accumulation to optimize osmotic regulation. Consequently, the allocation of organic carbon shifts towards moderating osmoregulation, leading to an increase in root-to-shoot ratios due to reduced carbon availability for shoot growth. Several studies have shown that plants under dehydration accumulate higher levels of fructose, glucose, sucrose, and raffinose compared to normal conditions (Fàbregas and Fernie 2019), aiding in the regulation of osmotic potential and mitigation of oxidative stress damages.

During periods of water shortage, plants tend to produce excessive reactive oxygen species (ROS), resulting in cellular damage to organelles and molecules (Zhang et al. 2020; Wahab et al. 2022). To counterbalance these detrimental effects, plants have evolved osmotic adjustment and antioxidant defense systems (Tariq et al. 2019a; Wahab et al. 2022). Enzymes such as peroxidase (POD), ascorbate peroxidase (APX), catalase (CAT), and superoxide dismutase (SOD) primarily eliminate ROS, enhancing stress resilience (Wahab et al. 2022). Furthermore, osmolytes like proline, glycine betaine, soluble proteins, and soluble sugars aid in maintaining cell turgidity, improving osmotic potential, enzyme activity, macromolecule protection, membrane stability, antioxidant mechanisms, and ROS detoxification, thereby promoting stress tolerance (Lukić et al. 2020; Ullah et al. 2022; Wahab et al. 2022). The diversion of assimilatory products to anti-stress mechanisms underlines why water deficit negatively impacts growth and biomass production.

Phytohormones play pivotal roles in normal and stressful conditions, orchestrating various physiological, biochemical, and molecular responses in plants, including drought tolerance (Iqbal et al. 2022; Ullah et al. 2022). Accordingly, drought increases abscisic acid (ABA) (Fleta-Soriano et al. 2015) and strigolactones (SLs) (Tariq et al. 2023) contribute significantly to drought tolerance. For instance, ABA regulates stress-responsive genes, osmolyte accumulation, dehydrin and LEA protein synthesis, and other stress-related proteins (Sreenivasulu et al. 2012). Drought-induced elevation in ABA levels contributes to the modulation of root growth and development, which is indispensable for ensuring sufficient water supply (Sharp et al. 2004). Moreover, Tariq et al. (2023) reported that strigolactones (SLs) modulate shoot and root architecture in nutrient-limited soil and facilitate P uptake by roots (Ruyter-Spira et al. 2011).

Furthermore, jasmonic acid (JA) and salicylic acid (SA) contribute to drought tolerance by regulating stomatal closure, ROS elimination, root growth, and enhancing cellular water conductivity (Iqbal et al. 2022). Salicylic acid (SA) accumulation has been reported to improve drought resistance (Miura and Tada 2014), by upregulating the antioxidant potential and protecting the photosynthetic apparatus from oxidative stress (Iqbal et al. 2022). Recent findings suggest that ethylene (ETH) stabilizes stress signals, enhancing plant adaptation to drought (Nazir et al. 2024). Moreover, these hormones, along with gibberellic acid (GA), indole acetic acid (IAA), and cytokinins (CTK), synergistically coordinate defense mechanisms (Iqbal et al. 2022; Rai et al. 2023) and promote growth and development, aiding plants in effectively coping with environmental stresses (Jiang and Asami 2018; Ullah et al. 2022).

Furthermore, the intricate interplay among hormones regulates the production of osmolytes, stomatal regulation, root growth, anti-oxidant potential, and other stress-responsive genes (Fleta-Soriano and Munné-Bosch 2016; Liu et al. 2022; Iqbal et al. 2022). Moreover, phytohormones also modulate photoassimilates, synthesis, distribution, and carbon balance in plants (Aksenova et al. 2012), with CK and ABA, for instance, regulating the source-sink relationship (Pospisilova et al. 2005). For example, CTK regulates growth and carbohydrate consumption and enhances starch biosynthesis by increasing the sink capacity (Ramawat and Merillon 2013). Thus, understanding the complex interactions among hormones and their regulation of antioxidant mechanisms, osmolyte production, and carbon balance is crucial for elucidating defense mechanisms under stress conditions.

A. sparsifolia Shap. (Camelthorn, family Fabaceae) is a perennial xerophytic shrub in central and eastern Asia. In China, this species is distributed throughout the Xinjiang Autonomous Region and Gansu, Inner Mongolia, and Qinghai provinces. In Xinjiang, it is primarily found in the Taklimakan Desert, which is hyperarid, saline, nutrient deficient, and water scarce. It has several ecological and social benefits including preventing desertification, dunes, and soil salinization and improving livelihoods (Tariq et al. 2022). Yet its abundance and habitat are threatened by rapid population growth, overgrazing, overharvesting, agricultural expansion, urbanization, and industrialization (Tariq et al. 2022, 2024). Its revegetation and restoration are thus urgently required. However, the availability of water is crucial for the growth and establishment of A. sparsifolia (Gui et al. 2013). Therefore, planting and establishing seedlings for revegetation and restoration in hyper-arid conditions are extremely challenging (Tariq et al. 2022). As a result, improving its seedling drought stress resilience is critical to its successful establishment.

N is vital for the growth, and overall function of plants. It plays various functions in the process of photosynthesis, respiration, storage, and transformation of photoassimilates, and many other activities (Vrede et al. 2004). A prolonged drought condition may severely hinder the mobility of N, resulting in a deficiency of N that ultimately limits tree growth (Mahieu et al. 2009). Trees are also impeded from metabolizing N by drought through disruption of enzyme activities associated with N metabolism. It’s a primary factor limiting the growth and productivity of trees and shrubs, particularly in arid regions where water scarcity further restricts their availability (Tariq et al. 2022). Clearly, this scenario illustrates a strong correlation between N availability and drought due to the severe restrictions imposed by drought on its accessibility and mineralization.

There is compelling evidence suggesting that the exogenous application of N plays a crucial role in enhancing the resilience of plants facing water deficit (Zhou et al. 2011; Tariq et al. 2019a; Zhang et al. 2020a, b) This improvement is attributed to enhancements in both enzymatic and non-enzymatic antioxidant systems, as well as regulation of photoassimilates and osmolytes (Tariq et al. 2019a; Li et al. 2020;). Additionally, N application facilitates the synthesis of proteins, and structural carbohydrates, leading to accelerated cell division and enhanced growth and biomass production (Huang et al. 2018). Moreover, the positive effects of N application on stress resistance and biomass production are associated with enhancements in hydraulic conductance (Xu et al. 2015b), N uptake and assimilation (Tariq et al. 2019a), cell wall elasticity, growth hormones, and increased meristematic tissues (Lawlor 2002).

In conditions of limited water availability, maintaining plant hydration is crucial for metabolic processes, and studies have indicated that leaf relative water content (LRWC) tends to increase following N addition, potentially due to the accumulation of osmolytes (Tariq et al. 2019a; Zhang et al. 2021a,b). The presence of N is indispensable for fundamental processes such as photosynthesis, respiration, and carbohydrate metabolism (Zhang et al. 2021a). There is a lack of comprehensive knowledge regarding how N addition affects sugar metabolism and its interplay with endogenous hormones and antioxidant mechanisms under conditions of water deficit. Furthermore, the majority of research has focused on non-woody plants, particularly crop species, with little attention given to understanding the effects of N application on drought resistance and metabolic alterations in shrubs like A. sparsifolia, which thrive in hyperarid, nutrient-deficient environments.

Previously, A. sparsifolia has been studied for its root’s transcriptomic analysis under drought (Wu et al. 2015), biomass and NP allocation patterns under drought, and N nutrition (Zhang et al. 2020). Plant adaptation is complex, so relying on limited indicators may be unreliable. It's crucial to assess multiple comprehensive indices to understand adaptation strategies. For instance, a plant's tolerance to environmental stress depends largely on its enzymatic and non-enzymatic antioxidant potential (Wahab et al. 2022), osmolytes production (Sami et al. 2016), and phytohormonal regulations (Fleta-Soriano et al. 2015; Fleta-Soriano and Munné-Bosch 2016; Neves et al. 2017; Iqbal et al. 2022). However, the physiological adaptation mechanisms of A. sparsifolia seedlings based on their sugar metabolism, phytohormones, and antioxidant potential have not yet been assessed under drought and N addition. Therefore, this study aimed to (a) determine the impact of drought on growth and biochemical changes in photosynthetic pigments, sugar metabolism, phytohormones, osmolytes, and antioxidant capacity in both leaves and roots of A. sparsifolia, and (b) decipher how N application affects these responses under water deficit.

2 Materials and Methods

2.1 Seedling Establishment

This study was conducted at the Cele National Station of Observation and Research for Desert-Grasslands Ecosystem at the southern fringe of the saline, nutrient-poor hyperarid Taklimakan Desert (37°00′N, 80°43′E). The area has a mean annual temperature of 11.9 °C, with extreme temperatures of 41.9 and −23.9 °C. The vegetation is sparse around oases, dominated by shrubs and sub-shrubs such as A. sparsifolia, Calligonum mongolicum, Karelinia caspica, and Tamarix ramosissima. The pot experiment was conducted in an outdoor nursery from May to September 2022. Healthy and viable seeds of A. sparsifolia were collected from the desert and planted in 42-L plastic pots (30 cm in diameter at the bottom, 35 cm in diameter at the top, 50 cm high, with a bottom hole) containing 38 kg of homogenized topsoil (0-30 cm) collected from around the study area. The soil was aeolian loamy sand with the following chemical properties (g kg−1): total N, 0.23; organic C, 2.99; total K, 23.11; total P, 0.60. Water was supplied to each pot (n = 1 seedling) on a three-day cycle to field capacity (18% w/w) during the first 30 days of the experiment.

2.2 Drought-Stress and N Application

One-month-old seedlings with uniform height were exposed to water deficit and N addition: (controlled, medium-drought (MD), and severe-drought (SD)] and N supplementation (0- and 4.0 gN⋅m−2 yr.−1). There were 12 replications of each treatment. The pots were maintained at a soil-relative water content (the ratio of soil water to water content at water-holding capacity) of 70–75% (well-watered, CK), 45–50% (medium drought), and 25–30% (severe drought) according to the following equation (Xu et al. 2009)

$$\mathrm{SRWC }= ([({{\text{W}}}_{{\text{soil}}} - {{\text{W}}}_{{\text{pot}}} -\mathrm{ D}{{\text{W}}}_{{\text{soil}}})/ ({{\text{W}}}_{{\text{FC}}} - {{\text{W}}}_{{\text{pot}}} -\mathrm{ D}{{\text{W}}}_{{\text{soil}}})] * 100)$$
(1)

Wsoil is the current soil weight (soil + pot + water), Wpot is the weight of the empty pot, DWsoil is the weight of dry soil, and WFC is the weight of soil at field capacity (soil + pot + water).

We rotated all pots every week to minimize positional effects. Moreover, a canopy made of transparent plastic film was used to cover the pots when it rained. The N application (pure urea 64%N) was applied once per month to the upper surface after watering. Five months fresh plants were harvested and separated into roots and leaves, which were wrapped in tinfoil, immediately immersed in liquid N, and stored at − 80 °C for physiological analysis.

2.3 Measurements of Relative Water Content (LRWC), Height, and Biomass

An electronic balance was used to measure the fresh weight (FW) of fully expanded leaves collected from each pot. The leaves were then soaked in distilled water for four hours at 4 °C in the dark. To determine the turgid weight (TW), the samples were weighed and transferred to an oven at 70 °C for 24 h before measuring their dry weight (DW). According to the following equation, the LRWC values were determined for each sample:

$$\mathrm{LRWC }= [(\mathrm{FW }-\mathrm{ DW})/(\mathrm{TW }-\mathrm{ DW})] \times 100\mathrm{\%}$$
(2)

Each plant was then split into above-ground (shoot) and below-ground (root), which were dried for 24 h at 70 °C before measurement of DW (g) using an electric balance.

2.4 Estimation of Concentrations of Photosynthetic Pigments

The concentrations of chlorophyll a (Chl a), and chlorophyll b (Chl b), in the fresh leaves were assessed using 80% acetone extracts following the method of Lichtenthaler and Wellburn (1983). We measured absorbances for Chl a, and Chl b at 663, and 646 respectively, using a U-1800 spectrophotometer (Hitachi, Tokyo, Japan).

2.5 Determination of Metabolites and Enzymes of Sugar Metabolism

The concentration of glucose and fructose was determined according to Johnson et al. (1964). Moreover, sucrose was determined using van Handel’s (1968) method. Citric acid was conducted using a standard method (Moellering and Gruber 1966). Starch was determined calorimetrically at 620 nm following a previous method (Hansen and Møller 1975).

The enzymatic activities of sugar metabolism were determined in leaf and roots extracts using appropriate ELISA kits following the manufacturer’s instructions (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China): SuSy (YJ302947), SPP (YJ550955), SPS (YJ302949), AMY (YJ190046), BAM (YJ190047), G6PDH (YJ513908), 6-PGDH (YJ241016), G6P (YJ550940), FrK (YJ364900), HK (YJ530492), PEPC (YJ224906) and Inv (YJ306942) (Sun et al. 2023).

2.6 Estimation of ROS and Malonaldehyde Concentrations

O2•– production rate was estimated by homogenizing samples (0.2 g) in phosphate buffer (pH 7.8) and centrifuging them at 5000 × g for 10 min. The reaction mixture consisting of 1 mL of supernatant, 0.1 mL of 10 mM hydroxylammonium chloride, and 0.9 mL of 65 mM phosphate buffer was used. A 20-min incubation at 25 °C was followed by the addition of 7 mM α-naphthylamine and 17 mM sulphanilamide. After adding ethyl ether, we centrifuged for five minutes at 1500 × g and measured the absorbance at 530 nm.

Hydrogen peroxide (H2O2) was determined by measuring the absorbance of the titanium-peroxide complex at 410 nm. The reaction mixture (supernatant: 1 mL, titanium reagent: 0.1 mL; ammonia: 0.2 mL), was centrifuged at 3000 × g for 10 min and washed with acetone and then centrifuged again at 10,000 × g for five minutes. In the next step, 1-M H2SO4 (3 mL) was added and the absorbance was read at 410 nm.

MDA was determined, following a standard method. Samples were homogenized with 5 ml of 1% trichloroacetic acid (TCA) and centrifuged at 5000 × g for 10 min. The supernatant was added with 205 TCA (4 mL, containing 0.5% thiobarbituric acid) and boiled for 30 min in a water bath at 95 °C. The reaction mixture was then cooled in an ice bath, and MDA was determined at reflectances of 450, 532, and 600 nm. MDA content was calculated using the following equation.

$$\mathrm{MDA }\left(\mathrm{mol g}-1\mathrm{ FW}\right)= 6.45\left({\text{OD}}532 -\mathrm{ OD}600\right)- 0.56{\text{OD}}450$$
(3)

2.7 Determination of Reduced and Oxidized Ascorbate and Glutathione and Antioxidant Enzyme Activities

The concentrations of ascorbate–glutathione metabolites and enzymatic activities were determined by rinsing 1.0 g of fresh samples of leaves and roots, freezing in liquid N, grinding to a powder, and mixing with 10 mL of 0.1 mol L−1 phosphate-buffered saline (PBS) (pH 7.0). The extracts were then centrifuged at 10 000 at 4 °C for 20 min to obtain supernatants for identifying markers of oxidative stress using appropriate ELISA kits following the manufacturer’s instructions (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China): SOD (YJ203409), CAT (YJ203416), POD (YJ203419), PPO (YJ203490), GPX (YJ203440), APX (YJ320415), GR (YJ203471), MDHAR (YJ103677), DHA (YJ880342), DHAR (YJ803460), AsA (mlsh0046), DHAR (YJ880342), GSSG (YJ302944), and GSH (YJ299073) (Liu et al. 2020; Li et al. 2022).

2.8 Determination of Concentrations of Endogenous Phytohormones

ELISAs were used to evaluate the concentrations of phytohormones. Briefly, frozen foliar and root samples were ground into powders with liquid N and combined with 5 mL of 0.1 mol L−1 PBS (pH 7.0). The concentrations of IAA (YJ803465), ZR (YJ803411), ABA (YJ803413), SLs (YJ803429), GA (YJ803421) BR (YJ803463), CT (YJ803406GA), and SA (YJ803461) were determined using appropriate ELISA kits following the manufacturer’s instructions (Shanghai Enzyme-linked Biotechnology Co., Ltd. Shanghai, China), respectively (Wu et al. 2020).

3 Statistical Analysis

An analysis of variance (ANOVA) in SPSS (Chicago, IL, United States) was used to test treatment differences in biomass, photosynthetic pigments, sugar metabolism, and antioxidant mechanisms. Means were compared using Duncan’s multiple range tests (p > 0.05). The figure graphics were created using GraphPad Prism 8. OriginPro (Version 2024, OriginLab Corporation, Northampton, MA, USA) was used to perform Pearson correlation analyses of growth characteristics, photosynthetic pigments, sugar metabolism, phytohormones, and antioxidants. Principal component analysis (PCA) of standardized data collected using OriginPro (Version 2024, OriginLab Corporation, Northampton, Ma, USA) was used to determine the overall complexity of drought and N response and to identify traits showing similar trends. With the help of this analysis, we were able to investigate the physiological responses to stress and integrate all responses to the drought stress and N treatment in leaves and roots.

4 Results

4.1 Changes in Growth and Chlorophyll Concentration

Under MD, the seedling height, shoot dry weight root dry weight, and leaf relative water content (LRWC) were reduced by 27, 36, 19, and 15%, respectively. Under SD, the reductions were 47, 51, 32, and 24% respectively. In contrast, the root/shoot ratio (RSR) increased up to 1.34 under MD and 1.40-fold under SD stress.

However, N application considerably reduced the drastic effects of drought stress and improved LRWC, plant height, shoot dry weight, and root dry weight by 1.20, 1.29, 1.35, and 1.12-fold under MD and by 1.12, 1.08, 1.15, and 1.13-fold under SD stress. However, the N application reduced the RSR under MD and had no significant impact under SD stress (Table 1).

Table 1 Changes in growth and chlorophyll pigments under drought and nitrogen addition
Full size table

The concentration of Chl a and Chl b fell by 31, 22% under MD, and 28 and 47% under SD stress. The ratio of Chl a/Chl b reduced by 11% under MD stress but increased up to 1.4-fold under SD stress. However, the N application enhanced Chl a up to 13 and 1.20 folds under MD and SD stress but had little effect on Chl-b and the ratio of Chl-/Chl-b (Table 1).

4.2 Sugar Metabolism

Under MD, the leaf concentrations of glucose increased 1.45 fold, and fructose 120 fold while the root concentrations increased 1.13 and 1.10 folds, respectively. Under SD, the increases were 1.26 and 1.27 folds in the leaves but only fructose increased (1.37 fold) in the roots. Conversely, sucrose and starch decreased by 16 and 22% in the leaves and by 34 and 15% in the roots under MD stress. The SD stress reduced their concentrations by 35- and 34% in the leaves and by 30 and 22% in the root (Tables 2 and 3). Glucose-6-phosphate (G6P) and citric acid (CA) in the leaves increased by 1.40, and 1.27 folds under MD, and by 1.29, and 1.21 folds under SD stress. In roots, CA increased significantly under MD (1.19 fold) stress, and G-6-P showed little changes under both stress (Tables 2 and 3).

Table 2 Changes in metabolites and enzymes of sugar metabolism in the leaves under drought and N addition
Full size table
Table 3 Metabolites and enzymes of sugar metabolism in the roots under drought and N addition
Full size table

Moreover, sucrose synthase (SuSy), α-amylase (AMY), and β-amylase (BAM) increased in the leaves by 1.33, 1.28 and 1.23 folds under MD and by 1.27, 1.12 and 1.19 folds under SD stress. In root, their concentration increased by 1.32, 1.32, and 1.25 folds under MD stress. Also, SD stress increased SuSy and AMY (1.14 and 1.29-fold) in roots but had little effect on BAM. Sucrose phosphate phosphatase (SPP) decreased by 19 and 30% in the leaves and 5 and 9% in the roots under MD and SD stress (Tables 2 and 3). Sucrose phosphate phosphatase (SPP) showed little changes in the leaves under both stresses but decreased in the roots by 19% under SD stress. Moreover, glucose-6-phosphate dehydrogenase (G6PDH), and hexokinase (HK) increased by 1.11 and 1.18 folds in the leaves and by 1.24 and 1.21 folds in the roots under MD stress. Their activities increased by 1.12 and 1.23 folds in the leaves and by 1.12 1.24 folds in the roots under SD stress. Also, 6-phosphogluconate dehydrogenase (6PGDH) enhanced in the leaves under both MD (1.19 fold) and SD stress (1.09 fold) but showed little changes in the roots. During drought fructokinase (FRK), and invertase (INV) showed little changes in both leaves and roots (Tables 2 and 3).

In leaves, N application increased glucose, fructose, G-6-P, and ST by 1.15, 1.17, 1.10, and 1.06 folds under MD and sucrose by 1.25 and 1.36 folds under both MD and SD stress. In roots, CA, fructose, and sucrose increased by 1.03, 1.27, and 1.43 folds under MD stress, ST by 1.18 folds under SD, and glucose by 1.25 and 1.33 folds under both MD and SD stress.

N application increased PEPC by 1.20 and 1.33 folds and FRK by 1.68 and 1.80 folds in the leaves under MD and SD stress. Also, SPP enhanced by 1.17 and 1.09 folds SPS by 1.12 and 1.09 folds in the roots and G-6-PDH increased in the leaves by 1.16 and 1.22 folds and in the roots by 1.12 and 1.08 folds under MD and SD stress. SPS increased in the leaves by 1.14-fold under MD and HK in the roots by 1.05-fold under SD in N-supplied seedlings (Tables 2 and 3). Additionally, N supplementation had little effects on INV and 6-P-GDH in both the leaves and roots and on SPP and HK in the leaves and FRK in the roots. Conversely, N addition reduced AMY in the leaves by 18 and 13% and in the roots by 15 and 7% under MD and SD stress. Also, SuSy reduced by 15 and 9% and BAM by 15 and 17% in the leaves under MD and SD stress. In roots, SuSy reduced by 11% under SD and BAM by 28% under MD stress (Tables 2 and 3).

4.3 Oxidative Stress Indicators and Anti-Oxidant Enzymes

MDA, O2•−, and H2O2 increased by 1.58, 1.38, and 1.45 folds in the leaves, and by 1.41, 1.18, and 1.02 folds in the roots under MD stress. Also, their concentrations increased by 2.03, 1.55, and 1.56 folds in the leaves and, 1.64, 1.24, and 1.14 folds in the roots under SD stress (Fig. 1a-c). However, N application reduced MDA, O2•−, and H2O2 in the leaves by 20, 12, and 17% under MD stress, and by 22, 25, and 172% under SD stress. A similar pattern was observed in the roots for MDA and O2•−, but had little effect on H2O2 (Fig. 1a-c).

Fig. 1
figure 1

Changes in the concentration of (a) superoxide anion (b) hydrogen peroxide (c) malondialdehyde, and enzyme activity of (d) superoxide dismutase (e) catalase (f) peroxidases (g) glutathione peroxidase, and (h) polyphenol oxidase under drought stress and N addition. Bars represent means ± SD (n = 3). different letters indicate treatment differences at p < 0.05 (Duncan’s method). The green and brown bars represent leaves and root samples. – N and + N stand for nitrogen applied and non-applied treatments. WW: well-watered; MD: medium drought; severe drought

Full size image

Leaves increased SOD by 1.21 and 1.18 folds, and CAT by 1.73 and 1.36 folds but reduced POD by 23 and 30% under MD and SD stress (Fig. 1d-f). GPX showed little changes under MD but increased by 1.09 fold under SD stress (Fig. 1g). Roots increased SOD, CAT, POD, and GPX under MD (1.34, 1.23, 1.06, and 1.24 folds) and SD stress (1.32, 1.39- and 1.17, and 1.34 folds). Conversely, PPO was reduced by 9 and 11% in the leaves and by 4 and 11% in the roots under MD and SD stress (Fig. 1h). N application increased CAT, POD, and SOD in leaves by 1.19, 1.19, and 1.26 folds under MD stress and by 1.28, 1.32, and 1.23 folds under SD stress. In roots, their activities increased by 1.21, 1.37, and 1.13 folds under MD stress. CAT and SOD increased by 1.24, and 1.11 folds under SD stress (Fig. 1d-f). However, N had little effect on GPX in the leaves and roots under both stress and PPO under MD stress. In root, N application increased PPO by 1.08 and 1.19 folds under both MD and SD stress (Fig. 1gh).

4.4 Responses of the Ascorbate–Glutathione Cycle to Drought Stress and N Addition

The concentrations of AsA decreased by 7 and 36% and GSH by 11- and 34% in the leaves under MD and SD stress. In roots, AsA under both stress and GSH under MD stress showed little changes. However, GSH reduced significantly in roots under SD stress (Fig. 2ab). Conversely, DHA and GSSG increased by 1.56 and 1.24 folds in the leaves and by 1.55 and 1.28 folds in the roots under MD stress. Also, their concentrations increased by 1.37 and 1.31 folds in the leaves and by 1.65 and 1.46 folds in the roots under SD stress (Fig. 2cd). Both MD and SD stress significantly increased APX, MDHAR in the leaves and roots, and GR in the roots (Fig. 3a-c). However, GR and DHAR were reduced in the leaves. In addition, SD stress reduced DHAR in the roots (Fig. 3d).

Fig. 2
figure 2

Changes in the concentration of (a) ascorbic acid, (b) glutathione, (c) dehydroascorbic acid (e) oxidized glutathione (e) ascorbic acid/dehydroascorbic acid ratio, and (f) glutathione/oxidized glutathione ratio under drought stress and N addition. Bars represent means ± SD (n = 3). different letters indicate treatment differences at p < 0.05 (Duncan’s method). The green and brown bars represent leaves and root samples. – N and + N stand for nitrogen applied and non-applied treatments. WW: well-watered; MD: medium drought; severe drought

Full size image
Fig. 3
figure 3

Changes in the enzyme activity of (a) ascorbate peroxidase (b) monodehydroascorbate reductase (c) glutathione reductase and (d) dehydroascorbate reductase under drought stress and N addition. Bars represent means ± SD (n = 3). different letters indicate treatment differences at p < 0.05 (Duncan’s method). The green and brown bars represent leaves and root samples. – N and + N stand for nitrogen applied and non-applied treatments. WW: well-watered; MD: medium drought; severe drought

Full size image

Compared to their non-treated peers, N application increased AsA by 1.19 and 1.32 folds in the leaves by 1.09 and 1.10 folds in the roots and GSH by 1.11 and 1.09 folds in the leaves under MD and SD stress. In roots, GSH increased under MD stress and showed little changes under SD stress. In contrast, N addition decreased DHA in the leaves by 16 and 6% and in the roots by 17 and 10% under MD and SD stress. Also, GSSG decreased by 17% in the leaves and by 12% in the roots under MD stress. N application significantly improved APX and DHAR in the leaves by 1.04 and 1.40 folds under MD, and by 1.27 and 1.32 folds under SD stress. In roots, their activities increased by 1.01 and 1.10 folds under MD, and by 1.18 and 1.19 folds under SD stress whereas GR increased in the leaves by 1.31 and 1.32 folds under both stresses. MDHAR increased in the leaves by 1.23 fold and in the roots by 1.33 folds and GR increased in the roots by 1.15 fold under MD stress. However, they showed little changes under SD stress supplied with N application (Fig. 3a-d).

4.5 Responses of Phytohormones Production to Drought Stress and N Addition

The concentrations of ABA, JA, SLs and increased significantly in both parts under MD and SD stress (Fig. 4a-c). Also, SA increased in the leaves under MD and SD and the roots under SD stress (Fig. 4d). Conversely, ZR and BR decreased in both parts under MD and SD stress (Fig. 4ef) and IAA, GA, and CTK decreased in the leaves under both drought stress (Fig. 5a-c). Moreover, IAA, GA, and CTK showed little changes in the roots under MD but reduced significantly under SD stress.

Fig. 4
figure 4

Changes in the concentration of (a) abscisic acid, (b) jasmonic acid (c) strigolactones (d) salicylic acid (e) zeatin riboside, and (f) brassinosteroids under drought stress and N addition. Bars represent means ± SD (n = 3). different letters indicate treatment differences at p < 0.05 (Duncan’s method). The green and brown bars represent leaves and root samples. – N and + N stand for nitrogen applied and non-applied treatments. WW: well-watered; MD: medium drought; severe drought

Full size image
Fig. 5
figure 5

Changes in the concentration of (a) indole acetic acid ratio, (b) gibberellic acid, and (c) cytokinin in leaf and root of A. sparsifolia seedlings in response to drought stress and nitrogen (N) supplementation. Bars represent means ± SD (n = 3). different letters indicate treatment differences at p < 0.05 (Duncan’s method). The green and brown bars represent leaves and root samples. – N and + N stand for nitrogen applied and non-applied treatments. WW: well-watered; MD: medium drought; severe drought

Full size image

N application increased ABA by 1.48 and 2.21 folds, SA by 1.12 and 1.109 folds, and GA by 1.08 and 1.03 folds in the leaves under MD and SD stress. Also, N addition increased GA in the roots in roots by 1.06 and 1.09 folds under MD and SD and ABA by 1.13 fold, and SA by 1.19 fold under MD stress (Figs. 4 and 5). CTK and BR increased in the leaves by 1.14 and 1.06 folds under MD and by 1.02 and 1.14 folds under SD stress. Further, IAA increased in the leaves by 1.15 fold under MD and in the roots by 1.06 and 1.10 folds under both MD and SD stress (Fig. 5). Meanwhile, ZR increased in the leaves and roots by 1.21 and 1.12 folds and SLs in the leaves by 1.25 fold under MD stress.

4.6 Relationships Between Growth and Physiology

We found that LRWC was positively correlated with shoot growth metrics (SDW and PH), chlorophyll pigments, the phytohormones (IAA, ZR, GA), and anti-oxidant mechanism (POD, DHAR, PPO, AsA, GSH, AsA/DHA, and GSH/GSSG), SPS, sucrose, INV while it was negatively correlated JA, ABA, SLs, G6P, SS, BAM, fructose, citrate, Chl-a/Chl-b and oxidative stress indicators (O2•−, H2O2, MDA, DHA, GSSG) (Fig. 5ab). RWM was positively correlated with sucrose metabolism (sucrose, SPSS, and SPS), AsA-GSH metabolites (GSH, AsA, and their ratios) and DHAR, as well as hormones (ZR, IAA, and GA), and negatively correlated with the oxidative stress (O2•−, H2O2, MDA, DHA, GSSG), antioxidant enzymes APX, GR, SOD, GPX, fructose, AMY, SS, PEPC, and RSR (Fig. 6cd).

Fig. 6
figure 6

Associations between growth metrics and stress tolerance indicators under drought and N addition (A) shoot growth and sugar metabolism, (B) shoot growth, hormones and antioxidants, (C) root growth and sugar metabolism, and (D) root growth, hormones and antioxidants

Full size image

4.7 Principle Component Analysis

The principal component analysis (PCA) was conducted to investigate the response of shoot and root sugar metabolism, hormones, and antioxidant enzymes (Fig. 7A-D) under varying levels of drought stress and N addition. The first two principal components (PCs) described 75% of the phytohormones and osmolytes variations in shoot under well-watered with nitrogen (WW + N) and severe drought (SD + N) with N (Fig. 7A). However, the PCA results of roots were 63% of the first two axes displaying that, SPP and FRK were negatively associated with BAM and AMY under SD (severe drought) stress (Fig. 7B). On the other hand, the PCA of antioxidants and hormones showed 84% of the first two axes in shoot and root 84% under drought and N treatments.

Fig. 7
figure 7

Principle component analysis (PCA) of responses of sugar metabolism, phytohormone and antioxidant indicators of stress tolerance under drought and N addition. A leaf sugar metabolism, B root sugar metabolism, C leaves hormones and antioxidants, and D roots hormones and antioxidants

Full size image

Specifically, in the shoot, phytohormones were closely correlated with each other under WW + N conditions, while antioxidant activities showed a positive correlation under SD + N conditions (Fig. 7c). Conversely, in roots, phytohormones exhibited a positive correlation with antioxidant activities under both SD + N and WW + N conditions. However, under SD condition, markers of oxidative stress such as MDA, DHA, GSSG, O2−, and H2O2 depicted a negative correlation with DHAR, GSH, IAA, and GA (Fig. 7D). Briefly, the PCA analysis unveiled a close and positive association between antioxidant enzymes, hormones, sugar accumulation, and enzymes of the pentose phosphate pathway under drought with N addition treatments (Fig. 7). Conversely, they exhibited a negative correlation with ROS, lipids peroxidation, and oxidized pools of GSH and GSSG, and sugar metabolism enzymes. This indicates that the enhanced antioxidant mechanisms, hormonal production, and coordinated regulation of sugar metabolism are pivotal factors exclusively contributing to the nitrogen-mediated enhancement of drought tolerance.

5 Discussion

5.1 Changes in Biomass and Chlorophyll Pigments

Drought stress reduced the LRWC and biomass production in A. sparsifolia seedlings, which is a common adverse effect of water deficit (Liu et al. 2015; Tariq et al. 2019a). Plant biomass is produced by the division and enlargement of cells, involving a complex interaction between physio-biochemical and molecular changes, which are susceptible to drought stress (Farooq et al. 2009). Thus, the growth response to drought in our study is likely to involve a variety of physiological and biochemical responses (Valdés et al. 2013). Water deficit reduces cellular turgidity and increases oxidative stress, thus impeding photosynthesis, which in turn reduces the supply of photoassimilates required for new cell growth, resulting in reduced biomass and growth (Tariq et al. 2019b; Li et al. 2020). Our correlation analysis indicates a strong negative correlation between oxidative stress markers (ROS, MDA, GSSG, DHA) with various growth and biomass parameters (Fig. 6). This suggests that the elevated levels of ROS, coupled with increased MDA levels, are associated with inhibited metabolism and impaired growth and biomass under drought stress (Gill and Tuteja 2010).

In our study, N addition increased the LRWC and biomass production. Adequate N supply promotes the synthesis of proteins and structural carbohydrates, leading to accelerated cell division, growth, and increased biomass production (Fig. 7) (Huang et al. 2018). The improved biomass production due to N supplementation has also been associated with enhancements in hydraulic conductance (Xu et al. 2015b), N uptake and assimilation (Tariq et al. 2019a), growth hormone levels, cell wall elasticity, and increased meristematic cell numbers (Lawlor 2002).

In conditions of low soil moisture, maintaining optimal plant-water relations is crucial for normal growth and metabolism. Previous studies have demonstrated that LRWC tends to increase following N application, likely attributed to the greater accumulation of osmolytes responsible for osmotic adjustment mechanisms (Tariq et al. 2019a; Zhang et al. 2021b). Osmolytes indirectly enhance LRWC by promoting improved water retention within leaf tissue, thereby maintaining cell turgor pressure. This mechanism ultimately contributes to the maintenance or increase of LRWC, particularly under drought conditions (Taiz and Zeiger 2010). The strong response of N in the hyperarid Taklimakan Desert supports the hypothesis that N application increases drought tolerance, thereby increasing hydration, biomass, and seedling survival. Similar effects of N have been reported in other arid ecosystems (Hooper and Johnson 1999; Zhou et al. 2011; Zhang et al. 2021b) as confirmed by our data.

Dry-land species may cope with their habitats by adapting to their morphologies (Patterson et al. 1997). In dry conditions, plants often reduce their growth rate and biomass production, making more biomass available to roots, to conserve water and increase water absorption. Prior studies on drought reported this phenomenon for desert species (Zhang et al. 2020; Ullah et al. 2022) which agrees with our findings. However, the N application significantly reduced the R/S ratio under MD stress. Generally, plants face a trade-off between protein allocation to photosynthesis and nutrient uptake (Patterson et al. 1997). According to Broadley et al. (Broadley et al. 2000), plants often use N primarily to produce and maintain leaves, and to optimize C fixation.

The increase in shoot biomass with N addition under MD stress in this study further supports this idea. Hence, N application enhanced shoot growth in Alhagi significantly under MD (Table 1), resulting in a decrease in the R/S ratio (Wu et al. 2008). Furthermore, our findings support the general theory that low soil nutrients lead to greater root growth (Tan and Hogan 1997), while high soil nutrients result in greater above-ground biomass partitioning (Li and Kang 2002).

5.2 Chlorophyll Concentration

In our study, drought stress significantly reduced the concentration of chlorophyll pigments which could be attributed to oxidative stress, reduced pigment synthesis, or increased chlorophyll degradation, all of which adversely affect photosynthesis and plant growth. There are previous reports of similar effects of drought on chlorophyll pigments in perennial xerophytes (Ullah et al. 2022; Zhang et al. 2021b, 2020). The concentration of photosynthetic pigments appears to be directly related to the concentration of N, which is an essential component of the photosynthetic apparatus (Farooq et al. 2009). Compared to non-applied, N application significantly increased Chl a under MD and SD stress (Table 1). Previous studies have shown that N-application promotes chlorophyll synthesis by increasing the concentration of proteins in stroma and thylakoids (Cooke et al. 2005) and the synthesis of chloroplasts during leaf development (Li et al. 2012) and ultimately improves net photosynthesis (Tariq et al. 2019a).

The ratio of Chl-a/Chl-b, serves as a valuable indicator to assess chlorophyll synthesis function, especially under stress conditions. In our study, we observed that the Chl-a/Chl-b ratios increased under SD stress regardless of N availability. This suggests that stress has a more pronounced impact on Chl-b compared to Chl-a (Gomes et al. 2017). The resulting elevation in the Chl-a/Chl-b ratio can be attributed to the preferential breakdown of Chl-b into Chl-a under stress conditions (Upadhyay and Panda 2005). Under stress, an increase in the Chl-a/Chl-b ratio enhances the plant's ability to capture and utilize light energy, thereby sustaining photosynthetic activity. Additionally, it promotes plant resilience against stress by optimizing the balance between light absorption and photoprotection (Nahakpam 2017). The adjustment in chlorophyll ratios is indicative of the plant's adaptive response to stress, which is intended to maintain essential physiological functions despite adverse environmental conditions.

5.3 Sugar Metabolism

A coordinated supply and utilization of carbon is critical for optimal plant growth and development under both normal and controlled conditions. As a result of this coordination, plants can efficiently utilize carbon resources to support essential physiological processes and accomplish optimal growth (Muller et al. 2011; Sami et al. 2016). Due to drought stress, photosynthetic products are inhibited and carbon balance is disrupted, resulting in growth reductions and physiological and metabolic disturbances (Du et al. 2020). Compared to well-watered seedlings, drought-stressed seedlings had higher glucose and fructose in both parts; N application increased glucose in leaves under MD stress and in roots under both stresses, while fructose levels increased in both parts under MD stress. Plants utilize soluble sugars in many ways in response to drought stress, including maintaining cell swelling pressure, and osmotic balance, preventing osmotic damage, and providing energy (Kaur et al. 2007; Sami et al. 2016).

Aside from serving as key substrates for energy metabolism sucrose, glucose, and fructose are also required to regulate signal transduction and stress responses during plant growth and development (Wingler and Roitsch 2008). Furthermore, under drought stress, plant cells often accumulate soluble sugars, which serve as osmoprotectants and energy sources. We suggest that N addition improves drought resistance of both species by increasing soluble sugar levels, as depicted in the PCA analysis (Fig. 7), where higher levels of soluble sugars are associated with drought stress and N addition (Van den Ende and Valluru 2009). In addition, sucrose and starch levels decreased in drought conditions, possibly as a result of an increase in the activities of enzymes responsible for degrading these compounds such as SuSy, AMY, and BAM. As illustrated in Fig. 6, there is a negative correlation between the activity of these enzymes and the level of these carbohydrates, which supports this inference. Previous studies have shown that drought stress increases the activity of sucrose metabolic enzymes to balance the sucrose level in cells (Du et al. 2020; Xu et al. 2015a). Additionally, the sugar status of a plant cell can down- or up-regulate enzymes that metabolize carbohydrates.

There was a significant increase in enzymes involved in sucrose metabolism under drought (Fig. 4A and B), suggesting that A. sparsifolia continuously improves its metabolic capacity under drought. Sucrose conversion into glucose and fructose is an effective way of improving osmotic regulation during drought. Sucrose is converted into glucose and fructose during glycolysis, which might explain the lower sucrose and higher glucose and fructose in our study. However, N application decreased the SuSy, resulting in sucrose accumulation. In plants, starch serves as an energy source and carbon storage compound, and it is affected by abiotic stresses such as drought and salinity (Sami et al. 2016; Thalmann et al. 2016). This study showed that drought stress decreased starch levels in both leaves and roots, which was in line with the upregulation of α-amylase and β-amylase activities in both parts. Starch degradation accelerated under drought stress by upregulating the expression of starch-degrading genes in Glycine max (GmAMY3 and GmBAM1) (Du et al. 2020) and Arabidopsis (Thalmann et al. 2016).

Starch degradation appeared to have accelerated in Alhagi, resulting in the conversion of starch into soluble sugars. However, nitrogen (N) addition has been reported to increase starch synthesis by regulating the expression of starch metabolism genes under drought (Lv et al. 2021) which agrees with our findings. The accumulation of starch and sucrose may indeed be correlated with lower activities of their degrading enzymes, as indicated by the negative correlation shown in Fig. 6. Further phosphorylation of the hexoses is carried out by HK and FRK (Renz and Stitt 1993). A recent study reported that the activity of HK and FRK decreases under drought stress (Shokat et al. 2020), which contradicts our findings. However, our results are in line with those of Whittaker et al. (2001), who noted that increased HK activity in Sporobolus stapfianus leaves might enhance drought tolerance. Fulda et al. (2011) reported that drought-tolerant sunflower plants under water deficit upregulated SlFRK3, a protein responsible for FK activity. Consequently, FRK increases following N supplementation appear to contribute to enhancing the drought resistance of A. sparsifolia seedlings.

Furthermore, G6PDH and 6PGDH increased in leaves under MD and SD stress, whereas in roots, G6PDH increased and 6PGDH remained unchanged. At both stress levels, N application further increased G6PDH in leaves and roots, as depicted by PCA analysis (Fig. 7). It is clear from our findings that stressed plants exhibit a high demand for the reduced form of NADPH, a demand that has continued to grow even after N application, satisfying a need for this nucleotide. The level of cytosolic G6PDH activity and transcripts may increase under stress to facilitate the synthesis of cofactors or intermediates related to tolerance mechanisms (Naliwajski and Skłodowska 2018). This enzyme controls the flow of carbon in the pentose phosphate pathway and produces NADPH, which is used in antioxidant pathways (Fig. 7) (Corpas and Barroso 2018), N metabolism, and amino acids synthesis (Sharkey and Weise 2016).

5.4 Changes in Oxidative Indicators and Antioxidant Enzymes

In plants under stress, ROS accumulation damages cell membranes and disrupts normal cell function, resulting in a reduction in growth (Gill and Tuteja 2010). In our study, significant ROS molecules (H2O2, and O2−) were detected under drought, regardless of N fertilization, which may represent higher lipid oxidation and changes in physiological responses resulting in lower growth, as corroborated by correlation analysis (Apel and Hirt 2004; Gill and Tuteja 2010; Tariq et al. 2018).

Antioxidant enzymes are produced by plants as a general mechanism to defend against drought stress, thereby preventing oxidative stress from damaging their tissues (Wahab et al. 2022). In our study, leaves exhibited significantly higher SOD and CAT in both MD and SD stress and GPX in SD-stressed seedlings. In roots, the activity of CAT, POD, and GPX increased at both stress levels. Several studies have shown that drought stress strengthens the antioxidant defense mechanisms in plants (Tariq et al. 2018; 2019a,b; 2022), which is consistent with our findings, suggesting that young A. seedlings can reduce drought-induced oxidative damage by upregulating the mechanisms of scavenging O2•− and H2O2 (Fig. 8).

Fig. 8
figure 8

Schematic representation of the interplay between leaf and root content of chlorophyll pigments, phytohormones, and sugar metabolism and antioxidant mechanism in A. sparsifolia seedlings under drought stress and nitrogen supplementation

Full size image

However, this was accompanied by a substantial decline in biomass production. In plants, nutrients are crucial for preventing adverse effects of drought, as they maintain enzyme activity, charge balance, and osmotic balance (Li et al. 2020). A PCA analysis showed that drought-stressed seedlings supplied with N had a greater positive correlation with antioxidant-enzyme levels compared to seedlings solely exposed to drought stress, indicating that N upregulates antioxidant potential (Fig. 7). For instance, N addition resulted in significantly higher CAT, POD, and SOD activity in leaves and roots under both stress levels. In roots, the PPO activity is upregulated under both MD and SD stress. Previous studies suggest that N application has no consistent impact on antioxidant enzyme regulation. According to our findings, N application under drought stress has a positive effect on antioxidant enzymes and likely improves membrane stability by reducing ROS accumulations (Figs. 7 and 8). Thus, N supply reduced oxidative damage to A. sparsifolia seedlings under drought stress, which agrees with the findings of previous studies (Zhong et al. 2017; Iqbal et al. 2020; Zhang et al. 2020).

In summary N application enhanced antioxidant enzyme activities and reduced ROS (H2O2, and O2−) and MDA levels, which indicates an increased redox defense system in response to drought (Fig. 7). In this regard, N application could improve drought resistance in A. sparsifolia seedlings through the production of antioxidant enzymes that help prevent cell membrane damage and promote effective dissipation of energy. In contrast, a recent study suggested that N application reduces antioxidant enzymes (Tariq et al. 2019a), which contradicts our findings. Perhaps these differences are a result of species-specific responses to N addition.

5.5 Changes in the AsA-GSH Cycle

AsA and GSH play a crucial role in scavenging ROS as part of the AsA-GSH cycle. Within this cycle, AsA is oxidized to MDHA by APX, and then further converted to DHA by removing H2O2. MDHA can be reduced back to AsA by MDHAR, and DHA can be regenerated into AsA through DHAR. Hence, the ratio of AsA/DHA serves as an indicator of the oxidation and reduction states within cells. In our study, MD and SD stress levels significantly AsA but increased DHA, resulting in a reduced ratio of AsA/DHA ratio. These findings are consistent with previous research (Jiang et al. 2022), suggesting that drought stress leads to decreased AsA and more oxidized AsA-GSH pools. This can be attributed to the increased activity of APX, which facilitated the H2O2 scavenging using AsA. Conversely, the observed decrease in DHAR activity may hinder the regeneration of AsA (Jiang et al. 2022).

The activities of APX, MDHAR, and DHAR are closely associated with increased stress tolerance (Jiang et al. 2022), while their low activity is linked to more severe membrane lipid peroxidation (Shao et al. 2008). N application significantly improved APX and DHAR in both parts and GR in leaves under both stress levels and MDHAR in leaves and roots under MD stress. Furthermore, GR upregulated in roots following N addition under MD stress. Hence, stabilizing the redox state of cells and increasing drought resistance (Jiang et al. 2022).

The ASA/DHA ratio, which represents the amount of ASA available for APX enzyme, increased in N-treated seedlings under drought stress. This may be attributed to the significant increase in MDHAR and DHAR activity. Moreover, the redox state of GSH influences the response of plants to stress, and the increase of GSH in this response determines the effectiveness of AsA. Adding N to A. sparsifolia restored the high GSH/GSSG ratio. Transforming GSSG to GSH enhances plants' ability to resist environmental stress (Verma et al. 2015). This mechanism may be a result of increased GR activity which increases GSH biosynthesis and reduces GSH degradation (Szalai et al. 2009), which is consistent with the findings in this study. Under stress, increased GR activity promotes H2O2 removal by GSH and maintains a high GSH/GSSG ratio (Szalai et al. 2009). It has been reported that H2O2 and GSH interact with stress signals in soybeans (Xing et al. 2020). This may explain why, in our experiment, N application reduced H2O2 by upregulating GR activity to increase the GSH and GSH/GSSG ratio.

The N application has therefore stabilized the redox state of A. sparsifolia cells by regulating the antioxidant enzymes and reducing H2O2 and MDA, resulting in improved drought resistance (Lukić et al. 2020). The PCA outcomes in our study, strongly suggest that N additions enhance drought tolerance in both species by bolstering their AsA-GSH cycle, leading to decreased H2O2 (Fig. 7). Several studies suggested that N application significantly reduces O2•− and H2O2 levels due to increased enzymatic and nonenzymatic antioxidant mechanisms (Chang et al. 2016; Tariq et al. 2019a; Zhang et al. 2021b). This reduction in oxidative stress contributes to enhanced membrane stability. Hence, our findings suggest that N application can substantially alleviate the detrimental effects of drought on membrane integrity and stability in A. sparsifolia cells.

5.6 Production of Phytohormones

In both normal and stressful conditions, phytohormones modulate several physiological processes. In our study, the concentration of ABA, JA, and SLs increased in leaves and roots under drought. In contrast, ZR decreased in both leaves and roots whereas IAA and GA decreased in leaves. Although IAA and GA did not exhibit significant changes in roots under MD, both declined under SD stress. These phytohormonal imbalances may cause marked growth and biomass reductions (Li et al. 2018). The growth features and ABA, JA, SA, and SLs were negatively correlated, suggesting that plants produced many anti-stress hormones to cope with drought (Fig. 6B, D). Given the interplay between hormones and drought, our findings suggest a potential strategy for coping with drought by adjusting the regulation of endogenous hormones (Li et al. 2018).

Phytohormones also modulate the response to oxidative stress by interacting with ROS and generating distinct transcriptomic and physiological responses resulting in plant acclimation. Plant respiratory burst oxidase homologs (RBOHs) play a critical role in this interaction. It has been shown that stress affects several hormones that act as regulators of ROS production and RBOH activity via various mechanisms (Devireddy et al. 2021) (Fig. 7). In drought, increasing ABA and JA modulate antioxidant mechanisms and osmolytes, thereby contributing to drought tolerance (Dhakarey et al. 2017; Iqbal et al. 2022). This confirms our findings, where a positive correlation was found between these hormones, antioxidant enzymes, and osmolytes (glucose and fructose) (Fig. 6). In addition, JA has been found to increase rapidly in Citrus (de Ollas et al. 2013) and Arabidopsis plants (Balbi and Devoto 2008). Drought-induced increases in ABA contribute to diverse mechanisms of stress tolerance including antioxidant activity and protection from photooxidative damage (Iqbal et al. 2022).

Also, GA has been reported to alter the regulatory functions of several genes within tomato plants exposed to drought, leading to reduced cell size, internode number, shoot length, and dry weight (Litvin et al. 2016). The interaction of drought with other hormones can also affect the activity, synthesis, metabolism, and transport of IAA in a variety of plants (Iqbal et al. 2022). Further, CTK concentration can increase or decrease under drought stress. Several physiological responses are regulated by CTK, including cell division, apical meristem support, and several physiological responses, which allow plants to respond and adapt to rapid environmental changes (Yadav et al. 2021). Growth hormones were negatively correlated with ROS and MDA (Fig. 6). Hence, their under drought could explain the sensitivity of plants to and severe decline in shoot and root growth and biomass, in our study.

N application significantly increased ABA in leaves under both stresses and JA and ABA in both leaves and roots under MD and SD. The increased ABA concentration, for instance, regulates a wide range of stress-related mechanisms (Danquah et al. 2014), for example, improvement in turgor pressure (Iqbal et al. 2022), and protects the xanthophyll cycle and the photosynthetic machinery from photooxidative stress (Zhu et al. 2011) as well as modulation of the osmotic and antioxidant potentials (Iqbal et al. 2022), leading to improved drought tolerance and growth responses. The N-treated MD-stressed seedlings had higher GA, IAA, and ZR in leaves; and IAA and GA in roots under MD and SD stress levels. Since both IAA and GA are principal growth stimulants in plants (Tiwari et al. 2017), higher concentrations under drought could result in increased shoot and root development and biomass in the N-fertilized A. sparsifolia seedlings.

Moreover, a growing body of evidence indicates that SLs, carotenoid-derived phytohormones, can play an important role in regulating plant response to environmental stress (Tariq et al. 2023). In our study, N application had no significant impact on SLs in roots under drought stress but increased in leaves under MD stress. However, regardless of N supply, SL concentration was still higher in seedlings. Dry soils are nutrient-limited particularly P availability, and drought further exacerbated their limitation (Tariq et al. 2022). There is evidence that SLs play an important role in modulating root and shoot development in response to nutrient deficiencies, particularly phosphorus, which promotes the synthesis of SLs (Andreo-Jimenez et al. 2015; Tariq et al. 2023). In Arabidopsis, Ruyter-Spira et al. (2011) reported that SLs enhance the growth of the lateral roots under limited P conditions by facilitating the uptake of P. Therefore, the upregulation of SLs in drought-stressed seedlings, particularly in roots, may have contributed to improved root growth and facilitated P uptake under drought. Therefore, SLs upregulation appears to be an important strategy for A. sparsifolia seedlings in the hyper-arid Taklamakan Desert to improve drought tolerance and facilitate root growth and nutrient uptake. Moreover, the PCA revealed that antioxidant enzymes, hormones, sugar accumulation, and enzymes of the pentose phosphate pathway were closely and positively associated with the drought and N addition treatments (Fig. 7), and negatively correlated with ROS, MDA, GSSG, and DHA and sugar degrading enzymes which suggests that enhanced antioxidant mechanisms, hormonal production and coordinated regulation of sugar metabolism exclusively confer the N-mediated improvement in drought tolerance.

6 Conclusion

Our study revealed that A. sparsifolia seedlings responded to N nutrition and performed better under drought stress, due to the coordinated regulations of hormones, sugar metabolism, and antioxidant mechanism. As compared to untreated seedlings, N supplementation increased the biomass and Chl-a production and improved the LRWC of drought-stressed seedlings. The N-mediated upregulation of H2O2 and O2-scavenging enzymes led to lower ROS and lipid peroxidation and improved AsA and GSH redox state. They also accumulated higher IAA, GA, CTK, BR, and ZR, which may account for their better growth. Hence, our findings provided evidence that N addition to drought-stressed seedlings promotes physiological and phytohormone response mechanisms that mitigate the effects of drought stress and this strategy could be adopted in programs for the restoration of hyperarid ecosystems. These findings have improved our understanding of the morpho-physiological plasticity of young xerophytes seedlings and their responses to N addition and water deficit before accessing groundwater resources, in the nutrient-deficient hyperarid desert–oasis ecotone of northwest China. However, likely variation in species responses to drought and nutrient supply necessitates further studies to determine the biochemical and molecular mechanisms of xerophyte seedlings for wider vegetation restoration and mitigation programs.