4.2. Responses of chlorophyll concentration to drought and N fertilization
The concentrations of chlorophyll a (Chl a), and chlorophyll b (Chl b) were significantly reduced under both drought conditions, irrespective of N availability, underscoring their sensitivity to drought conditions in xerophytic shrubs (Ullah et al. 2022b; Zhang et al. 2021, 2020). Photosynthetic pigments exhibit high sensitivity to water deficits, and their concentrations are directly linked to N availability because it is an integral structural component of photosynthetic machinery (Farooq et al. 2009). In response to drought, pigment concentrations may be reduced due to oxidative stress, chlorophyll degradation, reduced pigment synthesis, or nutrient availability, particularly N; all of which negatively impact photosynthesis and inhibit plant growth (Table 1). Chl-a and Chl-b levels were significantly improved by nitrogen fertilization under drought and well-watered conditions compared to non-fertilized peers (Table 1). According to previous research, nitrogen (N) promotes the formation of photosynthetic pigments by elevating protein levels in the stroma and thylakoids (Cooke et al. 2005) as well as chlorophyll synthesis during leaf development (Li et al. 2012) which subsequently increases net photosynthesis (Tariq et al. 2019a).
4.3. Responses of sugar metabolism to drought stress and N addition
In normal and stressful conditions, plant growth and development are dependent on coordinated carbon supply and utilization (Muller et al. 2011; Sami et al. 2016). Drought stress inhibits photoassimilates production and disrupts the carbon balance, resulting in physiological and metabolic alterations and biomass reduction (Du et al. 2020). In our study, the changes in carbohydrate composition, with enhanced fructose and sucrose and reduced starch, indicate a dynamic response of the Calligonum seedling's carbohydrate metabolism to either drought stress regime. Moreover, glucose increased in leaves under MD and in roots under SD may point to distinct regulatory mechanisms operating in leaves and roots under different stress intensities. Reduced starch levels may indicate mobilization of stored energy reserves to raise soluble sugar levels, possibly acting as osmoprotectants or energy sources (Rosa et al. 2009; Sami et al. 2016). Moreover, soluble sugar accumulation can protect cells by replacing the OH group with water, maintaining hydrophilic interactions between proteins and membranes, and preventing membrane damage under water deficit conditions (Hoekstra et al. 2001).
Moreover, it has been suggested that soluble sugar accumulation by plants (primarily sucrose) is associated with greater resistance to abiotic stresses (Van den Ende and Valluru 2009). Increased soluble sugar levels in our study may be the result of increased enzyme activities involved in sugar metabolism. For instance, the activities of AMY, BAM, and SPP upregulated in both leaves and roots while SuSy and Frk in leaves and INV in roots under either water stress (Table 3, 4). The upregulation of starch-degrading genes and the subsequent acceleration of starch degradation has been reported in Arabidopsis thaliana (Thalmann et al. 2016) and Glycine max (Du et al. 2020), which supports our findings. The observed changes in starch and soluble sugar levels (fructose, sucrose, glucose) and the corresponding alterations in the activity of sugar metabolism enzymes indicate a dynamic metabolic adjustment of Calligonum seedlings in response to water deficit conditions. The addition of N fertilization not only enhanced soluble sugar but also increased the accumulation of starch in Calligonum seedlings. This could be attributed to their metabolizing enzymes (Tables 2 and 3). Our results demonstrate that Calligonum seedlings are dependent on soluble sugar accumulation for osmotic balance, regardless of nitrogen availability.
HK and FRK further facilitate the phosphorylation of hexoses through additional enzymatic processes (Renz and Stitt 1993). According to a recent study, HK and FRK activity is reduced following drought stress (Shokat et al. 2020). In our study, FrK activity was significantly higher in leaves under either stress and HK under MD. In roots, their activities significantly decreased under SD stress compared to CK. This implies that the assimilating shoot and root demonstrate distinct responses in hexose phosphorylation under drought stress, reflecting the tissue-specific adaptation strategy of Calligonum to meet its metabolic needs during drought stress. Moreover, FRK in leaves and roots under MD, as well as HK in leaves under MD and in roots under either stress, exhibit significant upregulation following N supply. Fulda et al. (Fulda et al. 2011) suggested that drought-tolerant sunflower plants upregulated SlFRK3, an essential protein involved in FrK activity, under water deficit conditions. However, Whittaker et al., (Whittaker et al. 2001) reported that the higher activity of HK in Sporobolus stapfianus might confer drought tolerance. Hence, the observed increase in FrK and HK following N supplementation seems to play a role in conferring drought resistance to Calligonum seedlings.
As compared to CK, the enzymatic activity of G6PDH significantly increased in leaves and roots under either stress while that of 6PGDH leaves under SD. Upregulation of cytosolic G6PDH activity and transcripts has been suggested to be a cellular response to enhance NADPH production. This enzyme is responsible for controlling carbon flow in the pentose phosphate pathway and generating NADPH, a reducing equivalent that functions as an antioxidant to manage reactive oxygen species (ROS) and maintain redox balance and stress tolerance mechanisms (Dal Santo et al. 2012; Naliwajski and Skłodowska 2018) (Fig. 8). Moreover, it is clear that the leaves of stressed plants exhibit a high demand for the reduced form of NADPH; a demand that has been reduced under N fertilization in the form of decreased activity of G6PDH under either stress and 6PGDH under SD stress. Contrary to this, N-addition increased G6PDH and 6PGDH in roots under MD, thus satisfying the need for this nucleotide. Such responses of these enzymes to N addition might be associated with the N-induced alleviation of physiological responses.
4.4. Changes in oxidative indicators and antioxidant enzymes to drought and N fertilization
The measurement of MDA levels serves as a reliable biomarker for oxidative stress-induced damage to cell membranes in plants. In our study, both leaves and roots of Calligonum seedlings exhibited higher MDA levels under either stress, due to an increased accumulation of ROS (H2O2, and O2−) in both leaves and roots. Excessive ROS levels result in the degradation of proteins, lipids, and DNA, resulting in the death of cells (Apel and Hirt 2004). In addition to oxidizing the pigments, they may also elevate the concentrations of ABA which induces stomatal closure and therefore compromises photosynthesis and ultimately reduces biomass production (Carvalho 2008; Tariq et al. 2018). Both ASA and GSH are integral components of the cellular antioxidant system. They work together with enzymes of the AsA-GSH cycle to neutralize ROS and maintain cellular health (Shan et al. 2020). The redox buffering functions of AsA and GSH can trigger stress adaptation (Apel and Hirt 2004). In our study, DHA and GSSG led to reduced AsA and GSH in leaves and roots under either stress. Previous studies suggest that drought causes the AsA and GSH pools to decrease and become more oxidized (Jiang et al. 2022; Sarker and Oba 2018). Plants produce antioxidant enzymes in response to various environmental factors, including drought, to combat oxidative stress (Sunil et al. 2013; Tariq et al. 2019b; Ullah et al. 2022a).
The induction of antioxidant enzymes is a crucial process that plants employ to eliminate excessive ROS maintain cellular redox balance and reduce oxidative stress. The dynamic regulation of antioxidant enzyme activities in response to drought stress is a complex and highly fine-tuned process. In our study, both stress levels increased SOD, APX, CAT, and GPX in leaves under either stress and PPO in roots. Moreover, MD stress elevated CAT and GPX in roots, and POD in leaves. Previous studies reported that water deficit increases antioxidant defense mechanisms in trees and shrubs (Tariq et al. 2018, 2019a; Ullah et al. 2022b; Zhang et al. 2020), which is consistent with our findings. Under stress, antioxidant enzymes may decrease or increase, depending on the nature and duration of the stress exposure and the specific tissue of the plant. The variations observed between roots and assimilating shoots in our study suggest tissue-specific regulatory mechanisms. For example, APX and SOD decreased in roots but increased in assimilating shoot tissues; whereas PPO and POD decreased in assimilating shoots whereas in roots PPO enhanced but POD remained unchanged. This suggests that assimilating shoots and roots of Calligonum seedlings may imply a different strategy or reliance on alternative antioxidant systems to cope with oxidative stress.
The differential response between leaves and roots illustrates the complexity of the plant's overall stress adaptation strategy. The priorities of different organs may vary according to their specific functions and stress exposure (Du et al. 2020). The different responses of antioxidant enzymes to drought stress suggest a differential resource allocation or regulatory mechanisms of Calligonum seedlings to cope with oxidative stress. In challenging conditions, this organ-specific adaptation contributes to the plant's overall resilience and survival. In the AsA-GSH cycle, MDHAR, DHAR, and GR play pivotal roles in recycling and maintaining AsA and GSH pools. In our study, the reduced enzymatic activities of these enzymes suggest a compromised ability of Calligonum seedlings to regenerate and sustain the optimal concentration of AsA and GSH under drought conditions (Jiang et al. 2022).
In plants, an adequate nutrient supply is necessary for maintaining optimal enzyme activity, ensuring that essential metabolic pathways continue to function even under drought conditions (Li et al. 2020). Nitrogen application significantly upregulated SOD and CAT in leaves and roots, while POD and PPO in leaves under either stress. Moreover, PPO and POD in roots and GPX in leaves were significantly elevated by N addition upregulated MD stress. Our findings suggest that N supply improves the O2- and H2O2-scavenging ability of Calligonum seedlings by improving their enzymatic antioxidant mechanism, which helps the plants better cope with the oxidative stress associated with elevated levels of reactive oxygen species. Our findings are in line with previous studies on trees (Tariq et al. 2019a) and xerophytic shrubs (Zhou et al. 2011; Zhang et al. 2020). Studies have shown that high activities of APX, MDHAR, and DHAR are associated with greater tolerance to stress (Jiang et al. 2022). In contrast, their low activity leads to a greater degree of membrane lipid peroxidation (Shao et al. 2008).
A higher AsA/DHA ratio generally indicates a more reduced state and a better capacity for ROS scavenging. The N-mediated enhanced MDHAR and DHAR activities suggest an enhanced capacity of Calligonum seedlings to regenerate AsA from DHA, contributing to a higher AsA/DHA ratio, compared to unfertilized seedlings. In addition, GSH is critical for regenerating AsA and metabolizing H2O2 during the AsA-GSH cycle under normal and stressful conditions (Hojati et al. 2011). In stressful conditions, increased GR activity promotes the removal of H2O2, thereby maintaining a high GSH/GSSG ratio (Verma et al. 2015), which contradicts our findings. However, N supplementation restored the GSH concentration in Calligonum, which resulted in a high GSH/GSSG ratio. This could be attributed to increased GR activity which increases GSH biosynthesis and reduces its degradation (Szalai et al. 2009). High GSH/GSSG ratios indicate stress tolerance in plants. A transformation of GSSG into GSH enhances the ability of plants to resist environmental stresses (Verma et al. 2015).
Moreover, N addition has been reported to upregulate the APX activity for maintaining adequate H2O2 balance (Chang et al. 2016), which is in line with our results. Therefore, N fertilization contributes to the balancing of the ASA-GSH redox balance. This balance is crucial for maintaining the antioxidant defense system in plants, and its enhancement suggests an improved ability to counteract oxidative stress. Several studies have demonstrated that N fertilization significantly reduces the concentrations of O2•− and H2O2 as a result of the upregulation of both enzymatic and nonenzymatic antioxidant mechanisms in trees and xerophytes (Tariq et al. 2019a; Zhang et al. 2021, 2020), which results in lower oxidative stress and greater membrane stability. The coordinated mechanism observed in response to N fertilization suggests an adaptive response, enabling Calligonum seedlings to better cope with the oxidative stress associated with water deficit conditions.
3.6. Responses of phytohormones production to drought stress and N addition
As compared to CK, we observed significant increases in the concentration of ABA, JA, SA, and leaves and roots while SLs in roots under either stress. Furthermore, drought stress levels reduced IAA, CTK, and ZR in leaves and roots, as well as GA and BR in leaves. Phytohormones play crucial roles as signaling molecules, influencing various physiological mechanisms, growth, and development under normal and stressful conditions. Thus, the imbalance in hormone regulation might have resulted in a marked reduction in growth and biomass in our study (Li et al. 2018). Since hormone regulation is closely related to drought stress, our results may also be interpreted as a strategy to cope with drought through differential hormone regulation (Li et al. 2018). Additionally, phytohormones affect plant responses to oxidative stress through their interactions with ROS, resulting in distinct transcriptomic and physiological responses. This interaction is mediated by respiratory burst oxidase homologs (RBOHs) in plants. Multiple mechanisms have been demonstrated to influence the production of ROS and RBOH by stress (Devireddy et al. 2021) (Fig. 8). JA levels increase under drought conditions, modulating antioxidant mechanisms and osmolyte production, contributing to drought tolerance (Dhakarey et al. 2017). There is evidence that JA concentrations increase rapidly in Citrus (de Ollas et al. 2013) and Arabidopsis plants (Balbi and Devoto 2008), which supports our findings. Our study revealed that drought exposure increased ABA, which is involved in a wide variety of mechanisms for coping with stress, including antioxidant activity and the prevention of oxidative stress (Iqbal et al. 2022).
Compared to their unfertilized peers, N fertilization significantly improved ABA in leaves under either stress and in roots under MD stress. An increase in ABA concentration regulates several stress-related mechanisms (Danquah et al. 2014), including regulation of turgor pressure (Iqbal et al. 2022), protection of xanthophyll cycle and photosynthetic machinery (Du et al. 2010; Zhu et al. 2011), modulation antioxidant and osmotic potentials (Iqbal et al. 2022) leading to improved drought tolerance and growth responses.
Moreover, SA increased in leaves and roots under either stress. The SA accumulation improves drought resistance in plants by modulating several physiological responses including stomatal regulation, activation of defense responses, protection of photosynthetic machinery, and prevention of electron leakage (Iqbal et al. 2022). An increasing body of evidence suggests that SLs, which are carotenoid-derived phytohormones, can play a critical role in regulating plant response to stress conditions (Tariq et al. 2023). In our study, SLs increased in leaves under MD and in roots under either stress. The dry lands experience nutrient limitations, especially P and N availability, and the drought further aggravated the situation (Gao et al. 2022; Tariq et al. 2022). Indeed, there is evidence suggesting that SLs play a crucial role in modulating shoot-and-shoot architecture in response to nutrient limitations, which promotes the accumulation of SLs (Yoneyama et al. 2012; Andreo-Jimenez et al. 2015). Ruyter-Spira et al. (Ruyter-Spira et al. 2011) suggested that increasing SLs promotes lateral roots in a P-limited environment by facilitating P uptake. Hence, the upregulation of strigolactones in Calligonum seedlings appears to be a part of a multifaceted strategy to improve drought resilience, facilitate root growth, and improve nutrient uptake in the challenging conditions of the hyper-arid Taklamakan Desert. The increase in SL levels in response to N fertilization under SD conditions suggests a dynamic and adaptive response of Calligonum seedlings. SD conditions may have prevented seedlings from absorbing N optimally, which could have triggered the upregulation of SL biosynthesis.
Drought interaction with other hormones can also affect IAA activity, synthesis, metabolism, and transport in a variety of plants (Iqbal et al. 2022). Furthermore, GA has been reported to alter the regulation functions of several genes in tomato plants exposed to drought, resulting in smaller cells, fewer internodes, shorter shoots, and lower biomass (Litvin et al. 2016). Furthermore, CTK concentrations may increase or decrease in response to drought conditions. In addition to regulating cell division, CTK is involved in apical meristem support, and several physiological responses, which allow plants to adapt to rapid changes in the environment (Yadav et al. 2021). The observed decrease in the concentrations of IAA, GA, and CTK under drought stress in our study suggests that these hormonal imbalances could contribute to the sensitivity and severe reduction in shoot and root growth and biomass in Calligonum seedlings. In both leaves and roots, N fertilization increased IAAs and BR under MD while CTK and GA in leaves under MD and roots under SD. Moreover, ZR improved in leaves under either stress or roots under SD stress. Since these hormones play a role in stimulating plant growth (Tiwari et al. 2017); their increased concentration following N supplementation under drought might result in improved biomass of Calligonum seedlings compared to their non-fertilized peers.