Sodium nitroprusside and chitosan alleviate drought stress in spinach by modulating nutrient balance, phenolic production, and photosynthesis  

Sodium nitroprusside and chitosan alleviate drought stress in spinach by modulating nutrient balance, phenolic production, and photosynthesis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Sodium nitroprusside and chitosan alleviate drought stress in spinach by modulating nutrient balance, phenolic production, and photosynthesis Faezeh Khatami, Vahid Niknam This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9328377/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Drought perturbs water potential in the plants, led to oxidants accumulation and impaired cellular functions. The mineral nutrients are critical for adjusting water potential and modulating antioxidant activity and photosynthesis. The present study investigated the impact of sodium nitroprusside (SNP) and chitosan (CS) on six key mineral nutrients (Na, K, P, Ca, Mg, Fe) in spinach exposed to polyethylene glycol (PEG)-induced drought. The 3-leaved seedlings were irrigated with PEG (5%, 10%, and 15%) and treated by foliar spray of SNP (25 and 50 µM) and CS (15 and 30 mg L − 1 ). The physiological responses were studied by measuring the concentrations of hydrogen peroxide, malondialdehyde, chlorophylls, carotenoids, phenols, flavonoids, anthocyanins, and nutrients using UV/Vis spectroscopy and inductively coupled plasma optical emission spectrometry. Increasing drought intensity enhanced hydrogen peroxide accumulation and malondialdehyde. Drought stress led to higher Mg, while Fe remained constant due to its dual function in generating oxidants via Fenton reaction and stimulating antioxidants and photosynthesis. The SNP and CS application enhanced photosynthesis and alleviated the PEG-induced oxidative stress by enhanced production of phenolics and carotenoids. Both elicitors increased Ca, P and Fe contents and decreased Na, K and Mg contents. The intricate interplay between all six nutrients were critical to adjust water potential, minimize oxidative damage, and improve photosynthetic performance. Overall, the cross-talks between Fe, Mg and Ca were important for photosynthetic performance and antioxidant activity. The Na, K and P interplay was essential for osmotic adjustment. Biological sciences/Biochemistry Biological sciences/Physiology Biological sciences/Plant sciences drought mineral nutrients phenolics photosynthesis Spinacea Oleracea chitosan sodium nitroprusside Figures Figure 1 Figure 2 Figure 3 Introduction Spinach, Spinacea oleracea L., is a leafy green annual dicot herb belonging to Amaranthaceae family and Chenopodiaceae subfamily 1 . This plant is native to central and western Asia and a popular edible vegetable (named Persian plant in written records 2 ). Spinach possesses micro- and macro-nutrients essential for healthy human consumption including dietary fibers, vitamins (B, K, C), antioxidants, phenolics (flavonoids, anthocyanins), minerals (Ca, Fe, Mg, K, Mn, Cu, P) and other health promoting compounds. It has known for its therapeutic benefits because of anti-oxidant, anti-hypertension, anti-inflammatory, anti-aging and anti-bacterial activities 1 , 3 . The annual production of spinach in Iran is estimated ~ 101k tons based on FAO statistics 1 with an average yield of ~ 22 tons per ha, the world’s average yield is ~ 30 tons per ha 2 . Drought stress has imposed adverse effects on agricultural production, especially in countries such as Iran with ~ 88% arid and semi-arid climates 4 . To mitigate the adverse effects of drought stress, it is critical to understand the mechanisms of plant adaptation to drought and then apply effective drought management practices. Drought significantly changes the water potential and mineral composition surrounding the roots which significantly affects osmotic uptake of water. This phenomenon perturbs the osmolality and induce abscisic acid, leading to stomata closure and metabolic impairment. Stomata closure is the first reaction of most plants to reduce transpiration and conserve water, which in turn decrease CO 2 uptake, NADP + regeneration by the Calvin Cycle, and the photochemical activities 5 . Accordingly, it results in excessive accumulation of reactive oxygen species (ROS) and consequently severe oxidative damages to cellular components 6 . Photoinhibition and photo-destruction of photosynthetic pigments are other consequences of oxidative stress 7 . Oxidative damages to lipids, proteins, nucleic acids, photosynthetic pigments and enzymes disrupt normal cell functions 8 . The plant cells developed several adaptive processes to tolerate the drought depending on the growth stage, age and species of the plant as well as the intensity and duration of drought 9 . Two critical cellular processes for plant adaptation are photosynthesis and osmotic adjustment 10 . The photosynthetic apparatus generates the chemical energy for antioxidant defense processes. It also generates secondary metabolites like carotenoids to quench ROS and stabilize photosynthetic complexes 11 . The osmotic adjustment is mainly conducted by plant vacuoles. Moreover, the plant vacuoles produce secondary metabolites and more importantly sequester and digest the damaged components and toxic compounds. The phenolic compounds ( e.g ., flavonoids and anthocyanins) are the most substantial groups of secondary metabolites and antioxidants, produced from the shikimate-phenylpropanoid biosynthetic pathway in the vacuoles 12 , 13 . The flavonoids and anthocyanins significantly increased in pea and graph berries under drought, enabled plants to mitigate oxidative and dehydration stresses 14 Flavonoids are low molecular weight compounds and efficient chain-breaking antioxidants that can inhibit lipid peroxidation and reduce oxidative damages. Additionally, flavonoids possess anti-cancer and anti-inflammatory activities, and also beneficial for cardiovascular health upon human consumption 15 . Spinach is a well-known rich source of flavonoids 16 . Anthocyanins, a subset of flavonoids, are colored water-soluble pigments. Anthocyanins, recognized biomarkers of abiotic stress, act as signaling molecules and ROS scavengers, protecting photosynthetic machinery from photooxidation under drought stress 17 . The vacuole operation is tightly regulated by transport of solutes across tonoplast. The principal solutes in vacuoles are sodium (Na + ), potassium (K + ), chloride (Cl − ), calcium (Ca 2+ ), phosphates (PO 4 2− ), magnesium (Mg 2+ ), sulfate (SO 4 2− ), and nitrates (NO 3 − ). The principal elements involved in the photosynthesis and defense processes are iron (Fe), zinc (Zn), manganese (Mn), copper (Cu), boron (B), and molybdenum (Mo). Under drought, the concentrations of N, K, Ca, Mg, Na and Cl will increase while those of P and Fe will decrease 18 . The macro- and micro-nutrients play significant roles in cell structure, cell wall extensibility, turgor-related processes, respiration, and transpiration 9 , 19 . For example, potassium regulates the opening and closing of stomata, and controls carbohydrate metabolizing enzymes to enhance the accumulation and translocation of such osmolytes as sucrose 20 . Phosphorous, a macronutrient, is an important structural and functional component of biological molecules such as nucleic acids, phospholipids, and vitamins. It also plays a critical role in biological energy transfer processes, synthesis and transport of carbohydrates, nitrate reduction in roots, nuclear division and growth, transpiration rate and stomatal conductance. Iron, an essential micronutrient, is co-factor of several enzymes and proteins in the electron transport chain (photosystem I, photosystem II, ferredoxins, and cytochromes), chlorophyll synthesis, and nitrogen assimilation (nitrate reductase). Magnesium, as a co-factor of chlorophylls, is essential for the light absorption, chlorophyll synthesis, and Rubisco activation. Adequate Mg levels in plants promote the plant growth, improve water use efficiency, reduce the production of ROS, and activate the mechanisms for drought stress tolerance 21 . Mg also has the capacity to form complexes with ADP or ATP, which are the true substrates of most enzymes using adenine nucleotides 22 . Ca acts as a second messenger in plant signal transduction, and mediates plant adaptation to drought by activating the plasma membrane ATPase enzyme and calcium-calmodulin 23 . To induce adaptive mechanisms in spinach, the exogenous application of sodium nitroprusside (SNP) and chitosan (CS) were reported as an effective drought management practice 1 . Chitosan has been used as a non-toxic, biodegradable, and biocompatible natural biopolymer for agricultural applications. It reduces the negative effects of drought through the stress transduction pathway and the use of secondary messengers. Chitosan foliar application prevents water loss by transpiration and maximizes water usage 24 . The SNP, as the most prevalent NO-releasing substance, mediates diverse signaling pathways involved in plant response to biotic and abiotic stresses. The antioxidant properties of NO are demonstrated by its ability to trigger antioxidant activities and reduce ROS 25 . Given the prominent role of mineral nutrients in several cellular functions, the current research aimed at investigating the impact of SNP, and CS on six principal nutrients (Fe, Ca, Mg, P, Na, and K) of spinach under PEG-induced drought. The profile of these nutrients can lead us to cross-talks between them and how this intricate interplay can improve the plant tolerance to drought through effective performance of photosynthetic and vacuolar systems. The physiological functions of these systems were studied by assessing the contents of photosynthetic pigments, total phenols, flavonoids, and anthocyanins at certain levels of PEG-induced drought, CS, and SNP. Results Oxidative status in the spinach plants The PEG-induced drought increased the H 2 O 2 contents and consistently raised the MDA level (Fig. 1 ). The highest drought intensity led to the highest oxidative status, almost 2-fold (Fig. 1 ). The CS and SNP applications to the plants caused a significant reduction in the H 2 O 2 and MDA contents of the plants (Fig. 1 ). Higher concentrations of CS and SNP were more effective in alleviating oxidative status. Both concentrations of CS and SNP showed identical trends on the H 2 O 2 and MDA profiles. Also, the CS and SNP had nearly similar effects on the oxidative status. At normal condition, the combined applications of elicitors synergistically decreased peroxide contents, while at drought conditions, the co-application was slightly more effective in reducing oxidants compared to those of the separate applications (Fig. 1 ). Under PEG0% and PEG5%, the plants treated by either elicitor had shown similar H 2 O 2 and MDA contents (Fig. 1 a). At ≥PEG10%, there was a strong enhancement (more than 2-fold) in the H 2 O 2 contents of the plants treated by elicitors. The lipid peroxidation was also surged in the plants treated with >PEG10% induced drought intensities. Photosynthetic pigments in the spinach plants The contents of chlorophyll a , b and total chlorophylls showed a decreasing trend with increasing PEG levels (Fig. 2 a- 2 c). However, the contents of carotenoids raised with stepwise increase in the PEG (Fig. 2 d). The separate and combined applications of both CS and SNP led to higher pigments than their respective controls under certain PEG treatments (Fig. 2 ). The highest chlorophylls were observed in the control plants co-treated with SNP (50 µM) and CS (30 mg L − 1 ), averaged 84% higher than the non-elicited ones. Higher treatment levels, higher pigments. The lowest chlorophylls were measured in the non-elicited plants under PEG15%. At the highest PEG15%, the CS application had no significant effect on the chlorophylls. On the contrary, the co-application of CS and SNP synergistically increased the contents of chlorophylls even at high PEG levels compared to the control. The contents of carotenoids showed an increasing trend with the raising levels of CS and SNP (Fig. 2 d). The highest carotenoids were measured in the plants co-treated with both elicitors under PEG15%, almost 84% higher than the non-elicited plants. Antioxidant pigments in the spinach plants Total phenols, flavonoids, and anthocyanins consistently enhanced with the increasing PEG levels, Fig. 3 . The exogenous application of CS and SNP induced the production of phenols, flavonoids, and anthocyanins. Higher levels of both elicitors and also their combined application resulted in higher levels of these antioxidants. Both CS and SNP had similar induction effects. The moderate levels of either elicitors (25 µM SNP or 15 mg L − 1 CS) led to nearly identical enhancements in the metabolites. The combined application had a synergistic effect, led to approximately two-fold enhancement compared to control (Fig. 3 ). The highest metabolite contents were observed in the spinach plants co-treated with 50 µM SNP and 30 mg L − 1 CS under PEG15% (Fig. 3 ). Mineral composition in the spinach plants Consisdering the consistent trend of both concentrations of elicitors on the oxidative status, photosynthesis, and phenolics (Fig. 1 – 3 ), the effects of CS and SNP on the nutrients were only studied at the highest levels of 30 mg L − 1 and 50 µM, respectively (Table 1 ). The PEG-induced drought significantly increased the contents of intracellular Na, K, and Mg, while there was a singificant decrease in the contents of Ca, P, and Fe (Table 1 ). There was almost 25% enhancement in the former elements only upon exposure to PEG15%. The Ca and P showed a ~ 27% reduction only upon exposure to the highest PEG15% treatment. Fe had the lowest reduction (6%) and remained almost constant (Table 1 ). Under SNP treatment, the contents of Na, K, and Mg first raised in the plants and then remained constant with increasing drought intensities. On the contrary, the contents of P first decreased and then remained constant (Table 1 ). The contents of Fe was almost contant with no significant changes. Under CS treatment, the contents of Na, K, and Mg monotonically increased with increasing drought intensities, while the contents of Ca and P monotonically decreased. A similar trend to CS application was observed upon co-application of both elicitors. The CS was more potent than SNP in decreasing the contents of Na and Mg, while the SNP was more potent than CS in reducing the K content. The highest contents of Ca, P, and Fe and the lowest contents of Na, K, and Mg were observed in the control plants co-treated with both SNP (50 µM) and CS (30 mg L − 1 ) elicitors. The co-application of both CS and SNP had a synergistic effect on the Fe contents, with approximetly 5-fold increase compared to the plants under PEG-induced drought (Table 1 ). Table 1 Effects of CS (30 mg L − 1 ), SNP (50 µM), and PEG-induced drought on the concentrations (µg g − 1 DW) of mineral elements including Na, K, Ca, P, Mg, and Fe of Spinacia oleracea L. The values with similar letters are not significantly different at p ≤ 0.05. Results were expressed as means ± standard error (SE) Treatments Na K Ca P Mg Fe PEG 0% 1.13 d ±0.00006 4.71 d ±0.00007 1.67 l ±0.0003 0.33 f ±0.00007 0.88 d ±0.0003 0.0048 ij ±0.00009 PEG 5% 1.16 c ±0.00007 5.38 c ±0.00011 1.49 m ±0.00006 0.26 g ±0.00006 0.92 c ±0.00007 0.0045 j ±0.00005 PEG 10% 1.25 b ±0.00005 5.54 b ±0.00013 1.35 n ±0.00005 0.25 g ±0.00003 1.05 b ±0.00005 0.0045 j ±0.00007 PEG 15% 1.40 a ±0.00009 5.69 a ±0.00008 1.27 o ±0.00011 0.23 g ±0.00005 1.14 a ±0.0001 0.0045 j ±0.00005 PEG 0% + SNP 0.86 g ±0.00012 3.83 j ±0.00011 1.87 h ±0.00006 0.38 de ±0.00006 0.81 h ±0.0001 0.0066 g ±0.00006 PEG 5% + SNP 0.92 f ±0.00009 4.08 i ±0.00015 1.80 i ±0.0001 0.34 ef ±0.0001 0.85 ef ±0.00009 0.0055 h ±0.00007 PEG 10% + SNP 0.92 f ±0.00013 4.08 i ±0.00006 1.78 j ±0.0003 0.33 f ±0.0002 0.85 ef ±0.00006 0.0055 h ±0.00007 PEG 15% + SNP 0.94 e ±0.00011 4.08 i ±0.00006 1.75 k ±0.00009 0.33 f ±0.00006 0.87 de ±0.0001 0.0052 hi ±0.00007 PEG 0% + CS 0.74 j ±0.00012 4.10 h ±0.00011 1.97 e ±0.0001 0.42 ad ±0.0001 0.74 j ±0.00009 0.0096 e ±0.00006 PEG 5% + CS 0.80 i ±0.00011 4.50 g ±0.00005 1.95 f ±0.00006 0.41 bd ±0.0001 0.77 i ±0.00009 0.0093 e ±0.00004 PEG 10% + CS 0.82 h ±0.00007 4.51 f ±0.00011 1.93 g ±0.00005 0.40 bd ±0.00004 0.82 gh ±0.00007 0.0082 f ±0.00005 PEG 15% + CS 0.86 g ±0.00011 4.58 e ±0.00007 1.88 h ±0.0002 0.38 ce ±0.00005 0.84 fg ±0.00009 0.0071 g ±0.00008 PEG 0% + CS + SNP 0.62 n ±0.00011 3.25 n ±0.00011 2.24 a ±0.0001 0.46 a ±0.0001 0.50 n ±0.0001 0.0250 a ±0.00006 PEG 5% + CS + SNP 0.63 m ±0.00005 3.48 m ±0.00007 2.13 b ±0.0001 0.44 ab ±0.0001 0.53 m ±0.0001 0.0220 b ±0.0001 PEG 10% + CS + SNP 0.68 l ±0.00005 3.54 l ±0.00006 2.07 c ±0.0004 0.43 ab ±0.0003 0.61 l ±0.005 0.0180 c ±0.00006 PEG 15% + CS + SNP 0.72 k ±0.00005 3.72 k ±0.00007 1.98 d ±0.0001 0.42 ac ±0.00009 0.70 k ±0.0001 0.0140 d ±0.00005 Discussion Drought has a significant effect on water potential and mineral composition surrounding the roots. It will lead to excessive accumulation of ROS (Fig. 1 a) and consequently damage cellular components such as membranes (Fig. 1 b) and photosynthetic systems (Fig. 2 a- 2 c). The oxidative damage impairs the physiological functions or, at high intensities, induce the programmed cell death 26 . Lipid membranes and chloroplasts are highly vulnerable to oxidative damage under drought stress, leading to non-regulated transport across membranes, reduced chlorophyll rates and the activity of enzymes in the Calvin cycle during the photosynthesis 27 . Consistently, increasing drought accompanied with a decrease in chlorophylls and an increase in the total carotenoids (Fig. 2 ) and antioxidant phenolics (Fig. 3 ). Carotenoids have a protective role against ROS and protect chlorophyll against photooxidation 11 . Phenolics, in particular flavonoids and anthocyanins, are essential non-enzymatic antioxidants for alleviating drought stress. These major group of antioxidants play a key role in scavenging free radicals, stabilizing cell membranes, and preventing lipid peroxidation, thereby increasing drought resistance 12 . The contents of phenol, flavonoids, and anthocyanins in the plants elicited by either CS and SNP were almost equal in the plants under PEG 5% and PEG 0% (Fig. 3 ), in agreement with the contents of hydrogen peroxide at these conditions which were nearly equal as well (Fig. 1 a). Higher drought intensities significantly induced these secondary metabolites and antioxidants, led to a significant reduction in the MDA contents (Fig. 1 b). These physiological responses can be attributed to the significant changes in the nutrients of the plant cells. Under the adverse drought conditions, the plants alter the uptake of mineral elements to sustain cellular homeostasis and essential metabolic processes. Also, the interaction of minerals significantly affects the nutrient composition 28 . The exogenous application of SNP and CS significantly affected the nutrient uptakes through induction of osmo-protective agents and hormones, activation of ion-receptor genes, and regulation of oxidative status of the cells with notable consequences on plant physiology. Potassium and sodium, as macronutrients, are essential ions for osmotic adjustment and also key second messengers for diverse cellular processes 29 . K enhances relative leaf water content and water use efficiency under drought. Similarly, there were significant increases in the Na and K concentrations under drought mainly due to their key role in adjusting the water potential of the plant cells (Table 1 ). The accumulation of these inorganic ions in vacuoles is a cost-effective choice for plants under drought. The accumulation of Na ions changes the ionic balance. It seems that the use of chitosan, by reducing the amount of Na in the stem, has helped the plant tolerate drought stress. Besides, adequate K minimizes ROS generation by virtue of activating a series of antioxidant enzymes. Exogenous application of CS improves the moisture holding capacity of the plants and possibly contributed to lower concentrations of Na and K required for osmotic adjustment (Table 1 ). This property of CS can be attributed to its abscisic acid-dependent stomatal closure 24 . That is why, in agriculture, CS is mainly used for coating on seeds, leaves, and fruits. The application of CS oligomers was proposed to compensate the negative impact of drought stress on coffee, pepper, rice, corn, gerbera plants 30 . However, stomatal closure is accompanied by excessive accumulation of ROS 6 . Besides, CS was reported to stimulate the production of key osmotic regulators such as proline 16 and γ-aminobutyric acid 31 in drought stressed plants. Similarly, the exogenous application of nitric oxide can improve the relative water content in plants 32 , and consequently decrease the Na and K contents. Additionally, it triggers the vacuolar H + -ATPase activity which will increase the Na + /H + antiport activity 32 . As a result, the SNP application prevented the excessive accumulation of Na and the Na-mediated generation of oxidative stress (Table 1 and Fig. 1 ). Accordingly, SNP was more effective than CS in keeping the ion concentrations almost constant in the plants under increasing drought intensities (Table 1 ). Phosphorus makes the plants adaptive to water deficit by improving the stomatal conductance, photosynthetic rate, membrane stability, and water use efficiency 33 . High phosphorous contents inhibit the growth and development of roots, which can negatively influence the osmotic uptake of water and nutrients. Besides, high phosphorous concentrations disrupt photosynthesis (reduced Rubisco activity) and respirations. Lower phosphorous led to lower intracellular concentration of carbon dioxide. Considering the lower photosynthetic rate at higher drought intensities, the cells adjust the P intake with photosynthetic performance and intracellular CO 2 . Consistently, there was a decrease in phosphorous levels under drought stress (Table 1 ). CS treatment induces the adsorption of nutrients with negative charges into the leaves. In agreement with the present findings (Table 1 ), there were significant increase in the phosphorous of the spinach leaves treated with CS 34 . The CS and SNP both stimulate the antioxidant activity which will positively influence the P intake. As a result, the CS was more potent than SNP in increasing the P intake in the elicited plants (Table 1 ). Calcium, as a macronutrient and second messenger, is critical to maintain cell membrane structure and functionality. The calcium plays a key role in activating plasma membrane ATPase as well as Ca-calmodulin signaling for controlled metabolic activities. Consistently a drop in Ca under drought stress (Table 1 ) can be attributed to the ROS generation and cell membrane damage (Fig. 1 b). The CS and SNP application mediated the negative effects of oxidative stress, which possibly enhanced the Ca contents as it is required to supply the required nutrients lost during membrane damage. The simultaneous application of both elicitors was more effective than separate application, thus higher Ca than other cases (Table 1 ). The changes in Ca concentration under different treatments were consistent with the production of photosynthetic pigments and secondary metabolites (Figs. 2 and 3 ). Higher Ca concentration cause higher energy transport and antioxidant defense activity 35 . Consistently, the NO application improved the Ca uptake in wheat plants and activated the Ca-mediated antioxidant defense mechanism 32 . Besides, changes in Ca under these treatments can also be related to the K- and Na-induced Ca antagonism 36 . Under increasing drought intensity, the Mg as a macronutrient and co-factor of photosynthetic pigments, possibly increased to induce photosynthetic carbon metabolism. The exogenous application of NO induces the chlorophyll synthesis 32 , which is one possible reason for higher Mg content in the plants exposed only to SNP (Table 1 ). The Ca and Mg antagonism also affects their concentrations in the plants (Table 1 ). However, under drought, the changes in the concentration of Ca, as a competing cation, possibly resulted in a lower Mg concentration upon application of SNP and CS (Table 1 ). Under CS and SNP treatments, higher photosynthetic efficiency and higher antioxidant activity (Figs. 2 and 3 ) possibly minimized ROS accumulation and photosynthetic system disruption, obviated the need for excess magnesium to enhance photosynthetic CO 2 fixation 37 . The iron contents were not significantly changed with increasing drought intensity possibly because of two contradicting effects. Lower iron content increases the auxin synthesis, which is linked to the root development 28 . Higher root development led to higher osmotic water uptake. The Fe is an essential co-factor of antioxidant enzymes such as superoxide dismutase, catalase, and peroxidase 38 . As a result, the iron content will possibly increase with high drought levels. On the contrary, the excessive iron in the cells will stimulate ROS generation via Fenton reaction. As a result, the plants prefer lower iron contents under increasing drought as a possible defense mechanism to prevent ROS generation (Table 1 ). The K intake also impedes the absorption of iron 28 , in agreement with the present findings (Table 1 ). Higher K content decreases the Fe antagonistically. These two contradictory effects possibly led to almost constant Fe in the plants exposed to increasing drought intensities. Considering the recognized role of CS and SNP in inducing antioxidant enzyme activities, their application possibly improved iron uptake (Table 1 ) and also photosynthetic pigment biosynthesis (Figs. 2 and 3 ), as consistently reported in literature 16 , 31 . Consistently, the contents of chlorophylls surged upon co-application of SNP and CS due to both higher synthesis rate (Fig. 2 a- 2 c) at high iron contents and also less degradation rate at sharply reduced ROS (Fig. 1 a). The carotenoids and phenolics raised to quench singlet oxygen and to protect photosynthetic systems at elevated drought levels (Fig. 2 d). SNP stimulates mitochondrial aconitase (a component of the Krebs cycle) gene expression to IRP1 (iron regulatory protein) through increasing the plant’s access to iron 39 . Also, the NO application to the plants modulate the iron redox status and consequently increase the intracellular iron content 40 . Additionally, SNP directly increase the extracellular Fe content because of the chemical structure of SNP, Na 2 [Fe(CN) 5 NO].2H 2 O, which includes iron in Fe(II) form. As a result, the contents of Fe synergistically increased possibly because of high Fe demand. Further investigations on other mineral elements involved in the antioxidant enzymes and other essential defense pathways need to be investigated in the future. This understanding can lead us to modified culture media for the cultivations of plants in arid and semi-arid regions. Conclusion The CS and SNP effectively alleviated the PEG-induced drought stress in the spinach. The combined application of both elicitors at 30 mg CS L − 1 and 50 µM SNP showed the highest mitigating effects. Upon separate application of the elicitors and also at half-dose, a similar trend was observed in decreasing the oxidative stress and improving the production of photosynthetic pigments and phenolics. These treatments had significant effects on the profile of mineral nutrients. The contents of Na, K, and Mg increased and those of Ca, P, and Fe decreased under drought. The CS and SNP applications increased the contents Ca, P, and Fe and decreased the contents of Na, K, and Mg, while keeping the same trend of changes with drought intensity. The intricate interplay between these nutrients is critical to osmotic adjustment, antioxidant metabolism, and photosynthesis. Materials and Methods Plant materials and culture conditions Commercial Spinach seeds were bought from Pakan Bazr-e-Esfahan Co in Esfahan-Iran. The seeds were sterilized with sodium hypochlorite (1%) for 5 min, followed by thorough wash with double distilled water (dd H 2 O). The uniform sterilized seeds were germinated by placing them between two layers of filter paper in Petri dishes containing dd H 2 O. Until the emergence of radicles (~ 72 h), the dishes were kept in darkness at 25°C. The seedlings were then transferred to pots filled with peat and perlite (1:1) in a culture room, illuminated with light florescence lamps at an intensity of ~ 47 µmol m − 2 s − 1 under 16/8 h photoperiod and 25/18°C day/night temperature. The plants were grown in Hoagland culture media composed of 0.101 g L − 1 KNO 3 , 0.236 g L − 1 Ca(NO 3 ) 2 .4H 2 O, 0.115 g L − 1 NH 4 H 2 PO 4 , 0.246 g L − 1 MgSO 4 .7H 2 O, and 2 mL trace nutrients. The composition of micronutrients consisted of 1.864 g L − 1 KCl, 0.773 g L − 1 H 3 BO 3 , 0.169 g L − 1 MnSO 4 .H 2 O, 0.288 g L − 1 ZnSO 4 .7H 2 O, 0.062 g L − 1 CuSO 4 .5H 2 O, 0.040 g L − 1 H 2 MoO 4 . The iron was supplemented by 1 mL NaFeDTPA (30 g L − 1 , 10% Fe). The spinach plants at the three-leaf stage were treated by foliar spray of CS (15 and 30 mg L − 1 ), SNP (25 and 50 µM), and CS + SNP (15 + 25 and 30 + 50), under irrigation with four PEG concentrations (0, 5, 10 and 15%) after 1 d. These culture conditions were designed based on preliminary experimental works and literature review 1 . After 7 d, the samples were re-sprayed with the same elicitors. The plants undergone the PEG-induced drought for three weeks, and subsequently were harvested in the vegetative growth stage. The harvested plants were washed with dd H 2 O and then used for chemical assays immediately. The leaves were also double washed with dd H 2 O to remove any residual materials on the leaves and then dried in an air-circulated oven at 65°C for 24 h. The fresh weight to dry weight conversion factor was determined 5.79. Chemical assays Photosynthetic pigments The contents of photosynthetic pigments including chlorophyll a , b (Chl a , Chl b ) and carotenoids (C x+c ) were determined according to the standard method 41 . The fresh spinach leaves (0.5 g) were homogenized in acetone 80% and then centrifuged at 15,000×g for 30 min. The absorbance of the supernatant was read at the wavelengths of 646.8, 663.2, and 470 nm using a spectrophotometer (UV-Visible 160, Shimadzu, Japan). The contents of pigments were then estimated by the following empirical correlations 41 . Chl a (mg mL − 1 ) = 12.25 A 663.2 – 2.79 A 646.8 (1) Chl b (mg mL − 1 ) = 21.51 A 646.8 – 5.10 A 663.2 (2) C x + c (mg mL − 1 ) = (1000 A 470 – 1.82 Chl a – 85.02 Chl b ) /198 (3) Hydrogen Peroxide The contents of hydrogen peroxide were measured by the standard method 42 . The spinach leaves (0.1 g) were ground to homogenization in an ice bath with 5 mL TCA 0.1% w/v. The sample was centrifuged at 12,000 ×g for 20 min. A 0.5 mL aliquot of the supernatant was mixed with 0.5 mL phosphate buffer (10 mM, pH 7.0) and 1 mL KI (1 M). The solution absorbance was then read at 390 nm using the spectrophotometer. Malondialdehyde The contents of MDA, as a biomarker of lipid peroxidation, was measured using the standard method 43 . The spinach leaves (0.1 g) were homogenized in 5 mL TCA 0.1%w/v. The extract was centrifuged at 8000×g for 15 min. The supernatant (1 mL) was mixed with TCA/TBA reagent (4 mL) and the solution was heated at 95°C for 30 min and then cooled on ice immediately. The solution was centrifuged at 8000×g for 15 min. The supernatant’s absorbance was read 532 nm and the value was subtracted from the non-specific absorbance reading at 600 nm. The MDA contents were estimated using an extinction coefficient of 155 mM − 1 cm − 1 and expressed as mg g − 1 FW. Flavonoids The contents of flavonoids were determined by following the standard method in literature 44 . In brief, the spinach leaves (1 g) were ground to homogenization in acetic acid-ethanol (1:99 v/v) using a pre-chilled mortar and pestle. The tissue homogenate was centrifuged at 12,000×g and 4°C for 15 min. The supernatant was incubated in a water bath (80°C, 10 min) and then cooled down to room temperature. The sample absorbance was read at 270, 300, and 330 nm by the spectrophotometer. The concentration of flavonoids was estimated by the extinction coefficient of 33,000 mol – 1 cm – 1 , and expressed as µmol per g dry weight (DW). Anthocyanins The anthocyanin content was determined according to the standard method 45 . In brief, the harvested leaves (1 g) were homogenized in HCl-methanol solvent (1:99 v/v) using a pre-chilled mortar and pestle. The homogenate suspension was centrifuged at 12,000×g for 10 min. The supernatant was incubated in darkness at 4°C for 24 h, and then its absorbance was read at 550 nm by the spectrophotometer. The anthocyanin’s concentration was quantified by the extinction coefficient of 33,000 mol – 1 cm – 1 , and expressed as µmol per g leaf DW. Phenols Total phenol content was determined by the available method in literature 46 . In brief, the harvested leaves (1 g) were ground to homogenization in 1 mL dd H 2 O (80%) at 4°C, and centrifuged at 15,000×g for 15 min. A 100 µL aliquot of the diluted extract (1:20) was mixed with 50 µL Folin-Ciocalteu reagent (2 N), and the mixture was incubated at 25°C for 5 min. Thereafter, a 20% sodium carbonate solution (300 µL) was added to the reaction and the solution was further incubated at 25°C for 15 min. Ultimately, the sample was centrifuged at 12,000×g for 5 min, and the supernatant’s absorbance was read at 725 nm by the spectrophotometer. The total phenol was quantified by a calibration curve method using gallic acid as standard and expressed as µmol GAE per g leaf DW. Minerals The spinach leaves were washed with dd H 2 O and the sample was then dried in an air-circulated oven until reach to the constant weight. The minerals (Ca, Na, K, Fe, Mg, and P) were then extracted from the dried plants (0.5 g) by digestion in a mixture of concentrated nitric acid and perchloric acid (2:1v/v). Subsequently, the solution was analyzed using an inductively coupled plasma optical emission spectrometry (ICP-OES), Model VARIAN 725-ES, America, under optimized operating conditions. The concentrations of Fe, Mg, P, Na, K, and Ca were determined at the specific wavelengths of 261.2 nm, 279.8 nm, 213.6 nm, 589.6 nm, 766.5 nm, and 317.9 nm, respectively. Statistical analysis All the cultures and treatments of the spinach plants were done in triplicates. The plant leaves were sampled and used for further chemical assays. The means of the data were then analyzed by one-way analysis of variance in the SPSS Statistics Version 22.0 to identify statistically significant differences at p < 0.05. The results were presented as the mean ± standard deviation ( n = 3). Declarations Conflict of Interests The authors have no relevant financial or non-financial interests to disclose. Funding This work was supported by Iran National Science Foundation, Tehran-Iran, (Grant number 4014890). Author Contribution Both authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Faezeh Khatami. Supervision and data analysis was done by Vahid Niknam. The first draft of the manuscript was written by Faezeh Khatami. Both authors read and approved the final manuscript for submission. Acknowledgement Provision of the laboratory facilities by University of Tehran is greatly appreciated. We are thankful to the financial support by Iran National Science Foundation (Tehran, Iran) under grant number 4014890. Proof reading and editing by Hamyarapply Group (Tehran, Iran) is greatly appreciated. Data Availability All data generated or analyzed during this study are included in this article. References Khatami, F. & Niknam, V. Foliar application of sodium nitroprusside and chitosan induced antioxidant system and alleviated drought stress in spinach. Sci. Rep. 15 , 41636 (2025). Jonel, S., Natasa, K. & Biljana, G. V. Evaluation of profit and critical values in spinach production in the Republic of Serbia. Ekonomija 16 , 25–40 (2023). Nipa, N. A. et al. Growth, yield and biochemical qualities of spinach (Spinacia oleracea) being influenced by the foliar application of chitosan. J. Experimental Agric. Int. 45 , 30–40 (2023). Daneshvar, M. R. M., Ebrahimi, M. & Nejadsoleymani, H. An overview of climate change in Iran: Facts and statistics. Environmental Syst. Research 8 (2019). 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J., Chen, J. & Xiao, Q. & Verbruggen, N. An update on magnesium homeostasis mechanisms in plants Metallomics 5, 1170–1183 (2013). Wilkins, K. A., Matthus, E., Swarbreck, S. M. & Davies, J. M. Calcium-mediated abiotic stress signaling in roots. Front. Plant Sci. 7 , 1296 (2016). Jimenez-Gomez, C. P. & Cecilia, J. A. Chitosan: a natural biopolymer with a wide and varied range of applications Molecules 25 , 3981 (2020). Zhang, X., Qi, S., Liu, S., Mu, H. & Jiang, Y. Exogenous sodium nitroprusside alleviates drought stress in Lagenaria siceraria. Plants 13 , 1972 (2024). Ayala, A., Muñoz, F. & Argüelles, S. M. Lipid peroxidation: Production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxidative Medicine and Cellular Longevity 360438 (2014). (2014). Huseynova, I. M. Photosynthetic characteristics and enzymatic antioxidant capacity of leaves from wheat cultivars exposed to drought. Biochim. Biophys. Acta . 1817 , 1516–1523 (2012). Bhat, M. A. et al. Soil and mineral nutrients in plant health: A prospective study of iron and phosphorus in the growth and development of plants. Curr. Issues. Mol. Biol. 46 , 5194–5222 (2024). Visha Kumari, V. et al. Plant nutrition: an effective way to alleviate abiotic stress in agricultural crops. Int. J. Mol. Sci. 23 , 8519 (2022). Ghasemi Pirbalouti, A., Malekpoor, F., Salimi, A. & Golparvar, A. Exogenous application of chitosan on biochemical and physiological characteristics, phenolic content and antioxidant activity of two species of basil (Ocimum ciliatum and Ocimum basilicum) under reduced irrigation. Sci. Hort. 217 , 114–122 (2017). Rojas-Pirela, M., Carillo, P., La´rez-Vela´squez, C. & Romanazzi, G. Effects of chitosan on plant growth under stress conditions: similarities with plant growth promoting bacteria. Front. Plant Sci. 15 , 1423949 (2024). Alnusairi, G. S. H. et al. Exogenous nitric oxide reinforces photosynthetic efficiency, osmolyte, mineral uptake, antioxidant, expression of stress-responsive genes and ameliorates the effects of salinity stress in wheat. Plants 10 , 1693 (2021). Malhotra, H., Vandana, Sharma, S. & Pandey, R. in in Plant Nutrients and Abiotic Stress Tolerance . 171–190 (eds Hasanuzzaman, M.) (Springer Nature, 2018). Nipa, N. A. et al. Growth, yield and biochemical qualities of spinach (Spinacia oleracea) being influenced by the foliar application of chitosan. J. Experimental Agric. Int. 45 , 30–40 (2023). Khan, M. I. R. et al. Mineral nutrients in plants under changing environments: A road to future food and nutrition security. Plant. Genome . 16 , e20362 (2023). Ferreira, J. F. S., Filho, J. B., d., S., Liu, X. & Sandhu, D. Spinach plants favor the absorption of K+ over Na+ regardless of salinity, and may benefit from Na+ when K + is deficient in the soil. Plants 9 , 507 (2020). Waraich, E. A., Ahmad, R., Saifullah, M. Y. & Ehsanullah, A. Role of mineral nutrition in alleviation of drought stress in plants. Aust. J. Crop Sci. 5 , 764–777 (2011). Halliwell, B. Reactive species and antioxidants. Redox biology is a fundamental theme of aerobic life. Plant Physiol. 141 , 312–322 (2006). Rahikainen, M., Berkowitz, O., Whelan, J., Kangasjarvi, S. & Pascual, J. Role of aconitase in plant stress response and signaling. Physiol. Plant. 177 , e70128 (2025). Kumar, P., Tewari, R. K. & Sharma, P. N. Sodium nitroprusside-mediated alleviation of iron deficiency and modulation of antioxidant responses in maize plants. AoB Plants . plq002 , 1–11 (2010). Lichtenthaler, H. K. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol. 148 , 350–382 (1987). Velikova, V., Yordanov, I. & Edreva, A. Oxidative stress and some antioxidant systems in acid rain-treated bean plants: Protective role of exogenous polyamines. Plant Sci. 151 , 59–66 (2000). Heath, R. L. & Packer, L. Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125 , 189–198 (1968). Krizek, D. T., Kramer, G. F., Upadhyaya, A. & Mirecki, R. M. UV-B response of cucumber seedlings grown nder metal halide and high pressure sodium/deluxe lamps. Physiol. Plant. 88 , 350–358 (1993). Wagner, G. J. Content and vacuole/extravacuole distribution of neutral sugars, free amino acids, and anthocyanin in protoplasts. Plant Physiol. 64 , 88–93 (1979). Daley, J., Seevers, P. M. & Ludden, P. Studies on wheat stem rust resistance controlled at the Sr6 locus. III. Ethylene and disease reaction. Phytopathology 60 , 1648–1652 (1970). Footnotes FAO Stat 2024, https://data.un.org/data.aspx Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 04 May, 2026 Reviews received at journal 27 Apr, 2026 Reviews received at journal 25 Apr, 2026 Reviewers agreed at journal 16 Apr, 2026 Reviewers agreed at journal 15 Apr, 2026 Reviewers agreed at journal 14 Apr, 2026 Reviewers invited by journal 14 Apr, 2026 Editor invited by journal 13 Apr, 2026 Editor assigned by journal 07 Apr, 2026 Submission checks completed at journal 07 Apr, 2026 First submitted to journal 05 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9328377","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":625951334,"identity":"bdd60183-335b-42f9-a5f8-5452599224d9","order_by":0,"name":"Faezeh Khatami","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Faezeh","middleName":"","lastName":"Khatami","suffix":""},{"id":625951337,"identity":"69bc3a0e-587d-4926-b28d-9d8a03e3f613","order_by":1,"name":"Vahid Niknam","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtElEQVRIiWNgGAWjYDACZjYwJQflShCvxZiBjWgtUKWJDWzEusucnS3twc+cw+kb7jcwfvjBYJFPUItlM9txw95th3M3HGNgluxhkLBsIKTF4DB7mwTvtjSQFgZpoF8MCNoC0iL5d1taugHQlt9EamE7Js27zSYBqIWNWFvY0qRlt9kYzjyW2GbZY0CMlvPHzCTfbpOQ5zt8+PCNHxV1hLUgAcYGoAmkaBgFo2AUjIJRgBMAAJwRMXgcaDTwAAAAAElFTkSuQmCC","orcid":"","institution":"University of Tehran","correspondingAuthor":true,"prefix":"","firstName":"Vahid","middleName":"","lastName":"Niknam","suffix":""}],"badges":[],"createdAt":"2026-04-05 20:38:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9328377/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9328377/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107448199,"identity":"ce8fb1c1-4b8a-4a29-969b-831f6b65b312","added_by":"auto","created_at":"2026-04-21 14:56:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":125567,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CS, SNP, and PEG treatments on the contents of hydrogen peroxide (a) and malondialdehyde (b) in \u003cem\u003eSpinacia oleracea\u003c/em\u003e L. Values with similar letters are not significantly different at \u003cem\u003ep \u003c/em\u003e≤ 0.05. Vertical bars indicate mean ± SE (n=3).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9328377/v1/f3b628e27c6de6908904c763.png"},{"id":107448160,"identity":"7030c1ce-3a90-4021-8136-b59368372f29","added_by":"auto","created_at":"2026-04-21 14:56:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":803238,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CS, SNP, and PEG treatments on the chlorophyll \u003cem\u003ea\u003c/em\u003e (a), chlorophyll \u003cem\u003eb\u003c/em\u003e (b), total chlorophyll (c), and carotenoids (d) in \u003cem\u003eSpinacia oleracea\u003c/em\u003e L. Values with similar letters are not significantly different at \u003cem\u003ep \u003c/em\u003e≤ 0.05. Vertical bars indicate mean ± SE (n=3).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9328377/v1/fd2daf8729cfd286236175d4.png"},{"id":107448132,"identity":"2236567d-73c8-48ff-bb4c-fd1fe5ebdaba","added_by":"auto","created_at":"2026-04-21 14:56:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":406819,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CS, SNP, and PEG treatments on total phenol (a), flavonoids (b), and anthocyanin (c) of \u003cem\u003eSpinacia oleracea\u003c/em\u003e L. Values with similar letters are not significantly different at \u003cem\u003ep \u003c/em\u003e≤ 0.05. Vertical bars indicate mean ± SE (n=3).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9328377/v1/f338da07cae02f74e2ff9adf.png"},{"id":107448312,"identity":"24a30d9b-ded9-4fcc-92c6-c79fab05ec7a","added_by":"auto","created_at":"2026-04-21 14:57:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1722198,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9328377/v1/f9794479-6959-4432-ad3c-1cde56f1070e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sodium nitroprusside and chitosan alleviate drought stress in spinach by modulating nutrient balance, phenolic production, and photosynthesis ","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSpinach, \u003cem\u003eSpinacea oleracea\u003c/em\u003e L., is a leafy green annual dicot herb belonging to \u003cem\u003eAmaranthaceae\u003c/em\u003e family and \u003cem\u003eChenopodiaceae\u003c/em\u003e subfamily \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. This plant is native to central and western Asia and a popular edible vegetable (named Persian plant in written records \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e). Spinach possesses micro- and macro-nutrients essential for healthy human consumption including dietary fibers, vitamins (B, K, C), antioxidants, phenolics (flavonoids, anthocyanins), minerals (Ca, Fe, Mg, K, Mn, Cu, P) and other health promoting compounds. It has known for its therapeutic benefits because of anti-oxidant, anti-hypertension, anti-inflammatory, anti-aging and anti-bacterial activities \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe annual production of spinach in Iran is estimated ~\u0026thinsp;101k tons based on FAO statistics\u003csup\u003e1\u003c/sup\u003e with an average yield of ~\u0026thinsp;22 tons per ha, the world\u0026rsquo;s average yield is ~\u0026thinsp;30 tons per ha \u003csup\u003e2\u003c/sup\u003e. Drought stress has imposed adverse effects on agricultural production, especially in countries such as Iran with ~\u0026thinsp;88% arid and semi-arid climates \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. To mitigate the adverse effects of drought stress, it is critical to understand the mechanisms of plant adaptation to drought and then apply effective drought management practices.\u003c/p\u003e \u003cp\u003eDrought significantly changes the water potential and mineral composition surrounding the roots which significantly affects osmotic uptake of water. This phenomenon perturbs the osmolality and induce abscisic acid, leading to stomata closure and metabolic impairment. Stomata closure is the first reaction of most plants to reduce transpiration and conserve water, which in turn decrease CO\u003csub\u003e2\u003c/sub\u003e uptake, NADP\u003csup\u003e+\u003c/sup\u003e regeneration by the Calvin Cycle, and the photochemical activities \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Accordingly, it results in excessive accumulation of reactive oxygen species (ROS) and consequently severe oxidative damages to cellular components \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Photoinhibition and photo-destruction of photosynthetic pigments are other consequences of oxidative stress \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Oxidative damages to lipids, proteins, nucleic acids, photosynthetic pigments and enzymes disrupt normal cell functions \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe plant cells developed several adaptive processes to tolerate the drought depending on the growth stage, age and species of the plant as well as the intensity and duration of drought \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Two critical cellular processes for plant adaptation are photosynthesis and osmotic adjustment \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The photosynthetic apparatus generates the chemical energy for antioxidant defense processes. It also generates secondary metabolites like carotenoids to quench ROS and stabilize photosynthetic complexes \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The osmotic adjustment is mainly conducted by plant vacuoles. Moreover, the plant vacuoles produce secondary metabolites and more importantly sequester and digest the damaged components and toxic compounds. The phenolic compounds (\u003cem\u003ee.g\u003c/em\u003e., flavonoids and anthocyanins) are the most substantial groups of secondary metabolites and antioxidants, produced from the shikimate-phenylpropanoid biosynthetic pathway in the vacuoles \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The flavonoids and anthocyanins significantly increased in pea and graph berries under drought, enabled plants to mitigate oxidative and dehydration stresses \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eFlavonoids are low molecular weight compounds and efficient chain-breaking antioxidants that can inhibit lipid peroxidation and reduce oxidative damages. Additionally, flavonoids possess anti-cancer and anti-inflammatory activities, and also beneficial for cardiovascular health upon human consumption \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Spinach is a well-known rich source of flavonoids \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Anthocyanins, a subset of flavonoids, are colored water-soluble pigments. Anthocyanins, recognized biomarkers of abiotic stress, act as signaling molecules and ROS scavengers, protecting photosynthetic machinery from photooxidation under drought stress \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe vacuole operation is tightly regulated by transport of solutes across tonoplast. The principal solutes in vacuoles are sodium (Na\u003csup\u003e+\u003c/sup\u003e), potassium (K\u003csup\u003e+\u003c/sup\u003e), chloride (Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e), calcium (Ca\u003csup\u003e2+\u003c/sup\u003e), phosphates (PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e), magnesium (Mg\u003csup\u003e2+\u003c/sup\u003e), sulfate (SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e), and nitrates (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e). The principal elements involved in the photosynthesis and defense processes are iron (Fe), zinc (Zn), manganese (Mn), copper (Cu), boron (B), and molybdenum (Mo). Under drought, the concentrations of N, K, Ca, Mg, Na and Cl will increase while those of P and Fe will decrease \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe macro- and micro-nutrients play significant roles in cell structure, cell wall extensibility, turgor-related processes, respiration, and transpiration \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. For example, potassium regulates the opening and closing of stomata, and controls carbohydrate metabolizing enzymes to enhance the accumulation and translocation of such osmolytes as sucrose \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Phosphorous, a macronutrient, is an important structural and functional component of biological molecules such as nucleic acids, phospholipids, and vitamins. It also plays a critical role in biological energy transfer processes, synthesis and transport of carbohydrates, nitrate reduction in roots, nuclear division and growth, transpiration rate and stomatal conductance. Iron, an essential micronutrient, is co-factor of several enzymes and proteins in the electron transport chain (photosystem I, photosystem II, ferredoxins, and cytochromes), chlorophyll synthesis, and nitrogen assimilation (nitrate reductase). Magnesium, as a co-factor of chlorophylls, is essential for the light absorption, chlorophyll synthesis, and Rubisco activation. Adequate Mg levels in plants promote the plant growth, improve water use efficiency, reduce the production of ROS, and activate the mechanisms for drought stress tolerance \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Mg also has the capacity to form complexes with ADP or ATP, which are the true substrates of most enzymes using adenine nucleotides \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Ca acts as a second messenger in plant signal transduction, and mediates plant adaptation to drought by activating the plasma membrane ATPase enzyme and calcium-calmodulin \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo induce adaptive mechanisms in spinach, the exogenous application of sodium nitroprusside (SNP) and chitosan (CS) were reported as an effective drought management practice \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Chitosan has been used as a non-toxic, biodegradable, and biocompatible natural biopolymer for agricultural applications. It reduces the negative effects of drought through the stress transduction pathway and the use of secondary messengers. Chitosan foliar application prevents water loss by transpiration and maximizes water usage \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The SNP, as the most prevalent NO-releasing substance, mediates diverse signaling pathways involved in plant response to biotic and abiotic stresses. The antioxidant properties of NO are demonstrated by its ability to trigger antioxidant activities and reduce ROS \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGiven the prominent role of mineral nutrients in several cellular functions, the current research aimed at investigating the impact of SNP, and CS on six principal nutrients (Fe, Ca, Mg, P, Na, and K) of spinach under PEG-induced drought. The profile of these nutrients can lead us to cross-talks between them and how this intricate interplay can improve the plant tolerance to drought through effective performance of photosynthetic and vacuolar systems. The physiological functions of these systems were studied by assessing the contents of photosynthetic pigments, total phenols, flavonoids, and anthocyanins at certain levels of PEG-induced drought, CS, and SNP.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eOxidative status in the spinach plants\u003c/h2\u003e \u003cp\u003eThe PEG-induced drought increased the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e contents and consistently raised the MDA level (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The highest drought intensity led to the highest oxidative status, almost 2-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The CS and SNP applications to the plants caused a significant reduction in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and MDA contents of the plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Higher concentrations of CS and SNP were more effective in alleviating oxidative status. Both concentrations of CS and SNP showed identical trends on the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and MDA profiles. Also, the CS and SNP had nearly similar effects on the oxidative status. At normal condition, the combined applications of elicitors synergistically decreased peroxide contents, while at drought conditions, the co-application was slightly more effective in reducing oxidants compared to those of the separate applications (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUnder PEG0% and PEG5%, the plants treated by either elicitor had shown similar H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and MDA contents (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). At \u0026ge;PEG10%, there was a strong enhancement (more than 2-fold) in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e contents of the plants treated by elicitors. The lipid peroxidation was also surged in the plants treated with \u0026gt;PEG10% induced drought intensities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhotosynthetic pigments in the spinach plants\u003c/h3\u003e\n\u003cp\u003eThe contents of chlorophyll \u003cem\u003ea\u003c/em\u003e, \u003cem\u003eb\u003c/em\u003e and total chlorophylls showed a decreasing trend with increasing PEG levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). However, the contents of carotenoids raised with stepwise increase in the PEG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The separate and combined applications of both CS and SNP led to higher pigments than their respective controls under certain PEG treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The highest chlorophylls were observed in the control plants co-treated with SNP (50 \u0026micro;M) and CS (30 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), averaged 84% higher than the non-elicited ones. Higher treatment levels, higher pigments. The lowest chlorophylls were measured in the non-elicited plants under PEG15%. At the highest PEG15%, the CS application had no significant effect on the chlorophylls. On the contrary, the co-application of CS and SNP synergistically increased the contents of chlorophylls even at high PEG levels compared to the control.\u003c/p\u003e \u003cp\u003eThe contents of carotenoids showed an increasing trend with the raising levels of CS and SNP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The highest carotenoids were measured in the plants co-treated with both elicitors under PEG15%, almost 84% higher than the non-elicited plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAntioxidant pigments in the spinach plants\u003c/h3\u003e\n\u003cp\u003eTotal phenols, flavonoids, and anthocyanins consistently enhanced with the increasing PEG levels, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The exogenous application of CS and SNP induced the production of phenols, flavonoids, and anthocyanins. Higher levels of both elicitors and also their combined application resulted in higher levels of these antioxidants. Both CS and SNP had similar induction effects. The moderate levels of either elicitors (25 \u0026micro;M SNP or 15 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e CS) led to nearly identical enhancements in the metabolites. The combined application had a synergistic effect, led to approximately two-fold enhancement compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The highest metabolite contents were observed in the spinach plants co-treated with 50 \u0026micro;M SNP and 30 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e CS under PEG15% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eMineral composition in the spinach plants\u003c/h3\u003e\n\u003cp\u003eConsisdering the consistent trend of both concentrations of elicitors on the oxidative status, photosynthesis, and phenolics (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), the effects of CS and SNP on the nutrients were only studied at the highest levels of 30 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 50 \u0026micro;M, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe PEG-induced drought significantly increased the contents of intracellular Na, K, and Mg, while there was a singificant decrease in the contents of Ca, P, and Fe (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). There was almost 25% enhancement in the former elements only upon exposure to PEG15%. The Ca and P showed a\u0026thinsp;~\u0026thinsp;27% reduction only upon exposure to the highest PEG15% treatment. Fe had the lowest reduction (6%) and remained almost constant (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUnder SNP treatment, the contents of Na, K, and Mg first raised in the plants and then remained constant with increasing drought intensities. On the contrary, the contents of P first decreased and then remained constant (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The contents of Fe was almost contant with no significant changes. Under CS treatment, the contents of Na, K, and Mg monotonically increased with increasing drought intensities, while the contents of Ca and P monotonically decreased. A similar trend to CS application was observed upon co-application of both elicitors. The CS was more potent than SNP in decreasing the contents of Na and Mg, while the SNP was more potent than CS in reducing the K content. The highest contents of Ca, P, and Fe and the lowest contents of Na, K, and Mg were observed in the control plants co-treated with both SNP (50 \u0026micro;M) and CS (30 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) elicitors. The co-application of both CS and SNP had a synergistic effect on the Fe contents, with approximetly 5-fold increase compared to the plants under PEG-induced drought (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of CS (30 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), SNP (50 \u0026micro;M), and PEG-induced drought on the concentrations (\u0026micro;g g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW) of mineral elements including Na, K, Ca, P, Mg, and Fe of \u003cem\u003eSpinacia oleracea\u003c/em\u003e L. The values with similar letters are not significantly different at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05. Results were expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFe\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEG 0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.13\u003csup\u003ed\u003c/sup\u003e\u0026plusmn;0.00006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.71\u003csup\u003ed\u003c/sup\u003e\u0026plusmn;0.00007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.67\u003csup\u003el\u003c/sup\u003e\u0026plusmn;0.0003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.33\u003csup\u003ef\u003c/sup\u003e\u0026plusmn;0.00007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.88\u003csup\u003ed\u003c/sup\u003e\u0026plusmn;0.0003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0048\u003csup\u003eij\u003c/sup\u003e\u0026plusmn;0.00009\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEG 5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.16\u003csup\u003ec\u003c/sup\u003e\u0026plusmn;0.00007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.38\u003csup\u003ec\u003c/sup\u003e\u0026plusmn;0.00011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.49\u003csup\u003em\u003c/sup\u003e\u0026plusmn;0.00006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.26\u003csup\u003eg\u003c/sup\u003e\u0026plusmn;0.00006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.92\u003csup\u003ec\u003c/sup\u003e\u0026plusmn;0.00007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0045\u003csup\u003ej\u003c/sup\u003e\u0026plusmn;0.00005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEG 10%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.25\u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.00005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.54\u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.00013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.35\u003csup\u003en\u003c/sup\u003e\u0026plusmn;0.00005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.25\u003csup\u003eg\u003c/sup\u003e\u0026plusmn;0.00003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.05\u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.00005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0045\u003csup\u003ej\u003c/sup\u003e\u0026plusmn;0.00007\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEG 15%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.40\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.00009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.69\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.00008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.27\u003csup\u003eo\u003c/sup\u003e\u0026plusmn;0.00011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.23\u003csup\u003eg\u003c/sup\u003e\u0026plusmn;0.00005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.14\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0045\u003csup\u003ej\u003c/sup\u003e\u0026plusmn;0.00005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEG 0% + SNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.86\u003csup\u003eg\u003c/sup\u003e\u0026plusmn;0.00012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.83\u003csup\u003ej\u003c/sup\u003e\u0026plusmn;0.00011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.87\u003csup\u003eh\u003c/sup\u003e\u0026plusmn;0.00006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.38\u003csup\u003ede\u003c/sup\u003e\u0026plusmn;0.00006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.81\u003csup\u003eh\u003c/sup\u003e\u0026plusmn;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0066\u003csup\u003eg\u003c/sup\u003e\u0026plusmn;0.00006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEG 5% + SNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.92\u003csup\u003ef\u003c/sup\u003e\u0026plusmn;0.00009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.08\u003csup\u003ei\u003c/sup\u003e\u0026plusmn;0.00015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.80\u003csup\u003ei\u003c/sup\u003e\u0026plusmn;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.34\u003csup\u003eef\u003c/sup\u003e\u0026plusmn;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.85\u003csup\u003eef\u003c/sup\u003e\u0026plusmn;0.00009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0055\u003csup\u003eh\u003c/sup\u003e\u0026plusmn;0.00007\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEG 10% + SNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.92\u003csup\u003ef\u003c/sup\u003e\u0026plusmn;0.00013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.08\u003csup\u003ei\u003c/sup\u003e\u0026plusmn;0.00006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.78\u003csup\u003ej\u003c/sup\u003e\u0026plusmn;0.0003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.33\u003csup\u003ef\u003c/sup\u003e\u0026plusmn;0.0002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.85\u003csup\u003eef\u003c/sup\u003e\u0026plusmn;0.00006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0055\u003csup\u003eh\u003c/sup\u003e\u0026plusmn;0.00007\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEG 15% + SNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.94\u003csup\u003ee\u003c/sup\u003e\u0026plusmn;0.00011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.08\u003csup\u003ei\u003c/sup\u003e\u0026plusmn;0.00006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.75\u003csup\u003ek\u003c/sup\u003e\u0026plusmn;0.00009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.33\u003csup\u003ef\u003c/sup\u003e\u0026plusmn;0.00006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.87\u003csup\u003ede\u003c/sup\u003e\u0026plusmn;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0052\u003csup\u003ehi\u003c/sup\u003e\u0026plusmn;0.00007\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEG 0% + CS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.74\u003csup\u003ej\u003c/sup\u003e \u0026plusmn;0.00012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.10\u003csup\u003eh\u003c/sup\u003e\u0026plusmn;0.00011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.97\u003csup\u003ee\u003c/sup\u003e\u0026plusmn;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.42\u003csup\u003ead\u003c/sup\u003e\u0026plusmn;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.74\u003csup\u003ej\u003c/sup\u003e\u0026plusmn;0.00009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0096\u003csup\u003ee\u003c/sup\u003e\u0026plusmn;0.00006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEG 5% + CS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.80\u003csup\u003ei\u003c/sup\u003e\u0026plusmn;0.00011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.50\u003csup\u003eg\u003c/sup\u003e\u0026plusmn;0.00005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.95\u003csup\u003ef\u003c/sup\u003e\u0026plusmn;0.00006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.41\u003csup\u003ebd\u003c/sup\u003e\u0026plusmn;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.77\u003csup\u003ei\u003c/sup\u003e\u0026plusmn;0.00009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0093\u003csup\u003ee\u003c/sup\u003e\u0026plusmn;0.00004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEG 10% + CS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.82\u003csup\u003eh\u003c/sup\u003e\u0026plusmn;0.00007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.51\u003csup\u003ef\u003c/sup\u003e\u0026plusmn;0.00011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.93\u003csup\u003eg\u003c/sup\u003e\u0026plusmn;0.00005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.40\u003csup\u003ebd\u003c/sup\u003e\u0026plusmn;0.00004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.82\u003csup\u003egh\u003c/sup\u003e\u0026plusmn;0.00007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0082\u003csup\u003ef\u003c/sup\u003e\u0026plusmn;0.00005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEG 15% + CS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.86\u003csup\u003eg\u003c/sup\u003e\u0026plusmn;0.00011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.58\u003csup\u003ee\u003c/sup\u003e\u0026plusmn;0.00007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.88\u003csup\u003eh\u003c/sup\u003e\u0026plusmn;0.0002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.38\u003csup\u003ece\u003c/sup\u003e\u0026plusmn;0.00005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.84\u003csup\u003efg\u003c/sup\u003e\u0026plusmn;0.00009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0071\u003csup\u003eg\u003c/sup\u003e\u0026plusmn;0.00008\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEG 0% + CS\u0026thinsp;+\u0026thinsp;SNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.62\u003csup\u003en\u003c/sup\u003e\u0026plusmn;0.00011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.25\u003csup\u003en\u003c/sup\u003e\u0026plusmn;0.00011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.24\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.46\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.50\u003csup\u003en\u003c/sup\u003e\u0026plusmn;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0250\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.00006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEG 5% + CS\u0026thinsp;+\u0026thinsp;SNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.63\u003csup\u003em\u003c/sup\u003e\u0026plusmn;0.00005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.48\u003csup\u003em\u003c/sup\u003e\u0026plusmn;0.00007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.13\u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.44\u003csup\u003eab\u003c/sup\u003e\u0026plusmn;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.53\u003csup\u003em\u003c/sup\u003e\u0026plusmn;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0220\u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEG 10% + CS\u0026thinsp;+\u0026thinsp;SNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.68\u003csup\u003el\u003c/sup\u003e\u0026plusmn;0.00005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.54\u003csup\u003el\u003c/sup\u003e\u0026plusmn;0.00006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.07\u003csup\u003ec\u003c/sup\u003e\u0026plusmn;0.0004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.43\u003csup\u003eab\u003c/sup\u003e\u0026plusmn;0.0003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.61\u003csup\u003el\u003c/sup\u003e\u0026plusmn;0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0180\u003csup\u003ec\u003c/sup\u003e\u0026plusmn;0.00006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEG 15% + CS\u0026thinsp;+\u0026thinsp;SNP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.72\u003csup\u003ek\u003c/sup\u003e\u0026plusmn;0.00005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.72\u003csup\u003ek\u003c/sup\u003e\u0026plusmn;0.00007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.98\u003csup\u003ed\u003c/sup\u003e\u0026plusmn;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.42\u003csup\u003eac\u003c/sup\u003e\u0026plusmn;0.00009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.70\u003csup\u003ek\u003c/sup\u003e\u0026plusmn;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0140\u003csup\u003ed\u003c/sup\u003e\u0026plusmn;0.00005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDrought has a significant effect on water potential and mineral composition surrounding the roots. It will lead to excessive accumulation of ROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) and consequently damage cellular components such as membranes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) and photosynthetic systems (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The oxidative damage impairs the physiological functions or, at high intensities, induce the programmed cell death \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Lipid membranes and chloroplasts are highly vulnerable to oxidative damage under drought stress, leading to non-regulated transport across membranes, reduced chlorophyll rates and the activity of enzymes in the Calvin cycle during the photosynthesis \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Consistently, increasing drought accompanied with a decrease in chlorophylls and an increase in the total carotenoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and antioxidant phenolics (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Carotenoids have a protective role against ROS and protect chlorophyll against photooxidation \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Phenolics, in particular flavonoids and anthocyanins, are essential non-enzymatic antioxidants for alleviating drought stress. These major group of antioxidants play a key role in scavenging free radicals, stabilizing cell membranes, and preventing lipid peroxidation, thereby increasing drought resistance \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The contents of phenol, flavonoids, and anthocyanins in the plants elicited by either CS and SNP were almost equal in the plants under PEG 5% and PEG 0% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), in agreement with the contents of hydrogen peroxide at these conditions which were nearly equal as well (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Higher drought intensities significantly induced these secondary metabolites and antioxidants, led to a significant reduction in the MDA contents (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). These physiological responses can be attributed to the significant changes in the nutrients of the plant cells.\u003c/p\u003e \u003cp\u003eUnder the adverse drought conditions, the plants alter the uptake of mineral elements to sustain cellular homeostasis and essential metabolic processes. Also, the interaction of minerals significantly affects the nutrient composition \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The exogenous application of SNP and CS significantly affected the nutrient uptakes through induction of osmo-protective agents and hormones, activation of ion-receptor genes, and regulation of oxidative status of the cells with notable consequences on plant physiology.\u003c/p\u003e \u003cp\u003ePotassium and sodium, as macronutrients, are essential ions for osmotic adjustment and also key second messengers for diverse cellular processes \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. K enhances relative leaf water content and water use efficiency under drought. Similarly, there were significant increases in the Na and K concentrations under drought mainly due to their key role in adjusting the water potential of the plant cells (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The accumulation of these inorganic ions in vacuoles is a cost-effective choice for plants under drought. The accumulation of Na ions changes the ionic balance. It seems that the use of chitosan, by reducing the amount of Na in the stem, has helped the plant tolerate drought stress. Besides, adequate K minimizes ROS generation by virtue of activating a series of antioxidant enzymes.\u003c/p\u003e \u003cp\u003eExogenous application of CS improves the moisture holding capacity of the plants and possibly contributed to lower concentrations of Na and K required for osmotic adjustment (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This property of CS can be attributed to its abscisic acid-dependent stomatal closure \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. That is why, in agriculture, CS is mainly used for coating on seeds, leaves, and fruits. The application of CS oligomers was proposed to compensate the negative impact of drought stress on coffee, pepper, rice, corn, gerbera plants \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. However, stomatal closure is accompanied by excessive accumulation of ROS \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Besides, CS was reported to stimulate the production of key osmotic regulators such as proline \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and γ-aminobutyric acid \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e in drought stressed plants. Similarly, the exogenous application of nitric oxide can improve the relative water content in plants \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, and consequently decrease the Na and K contents. Additionally, it triggers the vacuolar H\u003csup\u003e+\u003c/sup\u003e-ATPase activity which will increase the Na\u003csup\u003e+\u003c/sup\u003e/H\u003csup\u003e+\u003c/sup\u003e antiport activity \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. As a result, the SNP application prevented the excessive accumulation of Na and the Na-mediated generation of oxidative stress (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Accordingly, SNP was more effective than CS in keeping the ion concentrations almost constant in the plants under increasing drought intensities (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePhosphorus makes the plants adaptive to water deficit by improving the stomatal conductance, photosynthetic rate, membrane stability, and water use efficiency \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. High phosphorous contents inhibit the growth and development of roots, which can negatively influence the osmotic uptake of water and nutrients. Besides, high phosphorous concentrations disrupt photosynthesis (reduced Rubisco activity) and respirations. Lower phosphorous led to lower intracellular concentration of carbon dioxide. Considering the lower photosynthetic rate at higher drought intensities, the cells adjust the P intake with photosynthetic performance and intracellular CO\u003csub\u003e2\u003c/sub\u003e. Consistently, there was a decrease in phosphorous levels under drought stress (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). CS treatment induces the adsorption of nutrients with negative charges into the leaves. In agreement with the present findings (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), there were significant increase in the phosphorous of the spinach leaves treated with CS \u003csup\u003e34\u003c/sup\u003e. The CS and SNP both stimulate the antioxidant activity which will positively influence the P intake. As a result, the CS was more potent than SNP in increasing the P intake in the elicited plants (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCalcium, as a macronutrient and second messenger, is critical to maintain cell membrane structure and functionality. The calcium plays a key role in activating plasma membrane ATPase as well as Ca-calmodulin signaling for controlled metabolic activities. Consistently a drop in Ca under drought stress (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) can be attributed to the ROS generation and cell membrane damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The CS and SNP application mediated the negative effects of oxidative stress, which possibly enhanced the Ca contents as it is required to supply the required nutrients lost during membrane damage. The simultaneous application of both elicitors was more effective than separate application, thus higher Ca than other cases (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The changes in Ca concentration under different treatments were consistent with the production of photosynthetic pigments and secondary metabolites (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Higher Ca concentration cause higher energy transport and antioxidant defense activity \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Consistently, the NO application improved the Ca uptake in wheat plants and activated the Ca-mediated antioxidant defense mechanism \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Besides, changes in Ca under these treatments can also be related to the K- and Na-induced Ca antagonism \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUnder increasing drought intensity, the Mg as a macronutrient and co-factor of photosynthetic pigments, possibly increased to induce photosynthetic carbon metabolism. The exogenous application of NO induces the chlorophyll synthesis \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, which is one possible reason for higher Mg content in the plants exposed only to SNP (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The Ca and Mg antagonism also affects their concentrations in the plants (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, under drought, the changes in the concentration of Ca, as a competing cation, possibly resulted in a lower Mg concentration upon application of SNP and CS (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Under CS and SNP treatments, higher photosynthetic efficiency and higher antioxidant activity (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) possibly minimized ROS accumulation and photosynthetic system disruption, obviated the need for excess magnesium to enhance photosynthetic CO\u003csub\u003e2\u003c/sub\u003e fixation \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe iron contents were not significantly changed with increasing drought intensity possibly because of two contradicting effects. Lower iron content increases the auxin synthesis, which is linked to the root development \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Higher root development led to higher osmotic water uptake. The Fe is an essential co-factor of antioxidant enzymes such as superoxide dismutase, catalase, and peroxidase \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. As a result, the iron content will possibly increase with high drought levels. On the contrary, the excessive iron in the cells will stimulate ROS generation via Fenton reaction. As a result, the plants prefer lower iron contents under increasing drought as a possible defense mechanism to prevent ROS generation (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The K intake also impedes the absorption of iron \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, in agreement with the present findings (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Higher K content decreases the Fe antagonistically. These two contradictory effects possibly led to almost constant Fe in the plants exposed to increasing drought intensities.\u003c/p\u003e \u003cp\u003eConsidering the recognized role of CS and SNP in inducing antioxidant enzyme activities, their application possibly improved iron uptake (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and also photosynthetic pigment biosynthesis (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), as consistently reported in literature \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Consistently, the contents of chlorophylls surged upon co-application of SNP and CS due to both higher synthesis rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) at high iron contents and also less degradation rate at sharply reduced ROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The carotenoids and phenolics raised to quench singlet oxygen and to protect photosynthetic systems at elevated drought levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). SNP stimulates mitochondrial aconitase (a component of the Krebs cycle) gene expression to IRP1 (iron regulatory protein) through increasing the plant\u0026rsquo;s access to iron \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Also, the NO application to the plants modulate the iron redox status and consequently increase the intracellular iron content \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Additionally, SNP directly increase the extracellular Fe content because of the chemical structure of SNP, Na\u003csub\u003e2\u003c/sub\u003e[Fe(CN)\u003csub\u003e5\u003c/sub\u003eNO].2H\u003csub\u003e2\u003c/sub\u003eO, which includes iron in Fe(II) form. As a result, the contents of Fe synergistically increased possibly because of high Fe demand.\u003c/p\u003e \u003cp\u003eFurther investigations on other mineral elements involved in the antioxidant enzymes and other essential defense pathways need to be investigated in the future. This understanding can lead us to modified culture media for the cultivations of plants in arid and semi-arid regions.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe CS and SNP effectively alleviated the PEG-induced drought stress in the spinach. The combined application of both elicitors at 30 mg CS L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 50 \u0026micro;M SNP showed the highest mitigating effects. Upon separate application of the elicitors and also at half-dose, a similar trend was observed in decreasing the oxidative stress and improving the production of photosynthetic pigments and phenolics. These treatments had significant effects on the profile of mineral nutrients. The contents of Na, K, and Mg increased and those of Ca, P, and Fe decreased under drought. The CS and SNP applications increased the contents Ca, P, and Fe and decreased the contents of Na, K, and Mg, while keeping the same trend of changes with drought intensity. The intricate interplay between these nutrients is critical to osmotic adjustment, antioxidant metabolism, and photosynthesis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and culture conditions\u003c/h2\u003e \u003cp\u003eCommercial Spinach seeds were bought from Pakan Bazr-e-Esfahan Co in Esfahan-Iran. The seeds were sterilized with sodium hypochlorite (1%) for 5 min, followed by thorough wash with double distilled water (dd H\u003csub\u003e2\u003c/sub\u003eO). The uniform sterilized seeds were germinated by placing them between two layers of filter paper in Petri dishes containing dd H\u003csub\u003e2\u003c/sub\u003eO. Until the emergence of radicles (~\u0026thinsp;72 h), the dishes were kept in darkness at 25\u0026deg;C. The seedlings were then transferred to pots filled with peat and perlite (1:1) in a culture room, illuminated with light florescence lamps at an intensity of ~\u0026thinsp;47 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under 16/8 h photoperiod and 25/18\u0026deg;C day/night temperature.\u003c/p\u003e \u003cp\u003eThe plants were grown in Hoagland culture media composed of 0.101 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KNO\u003csub\u003e3\u003c/sub\u003e, 0.236 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Ca(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e.4H\u003csub\u003e2\u003c/sub\u003eO, 0.115 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NH\u003csub\u003e4\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.246 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO, and 2 mL trace nutrients. The composition of micronutrients consisted of 1.864 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KCl, 0.773 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e, 0.169 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MnSO\u003csub\u003e4\u003c/sub\u003e.H\u003csub\u003e2\u003c/sub\u003eO, 0.288 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ZnSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO, 0.062 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e CuSO\u003csub\u003e4\u003c/sub\u003e.5H\u003csub\u003e2\u003c/sub\u003eO, 0.040 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e H\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e. The iron was supplemented by 1 mL NaFeDTPA (30 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 10% Fe).\u003c/p\u003e \u003cp\u003eThe spinach plants at the three-leaf stage were treated by foliar spray of CS (15 and 30 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), SNP (25 and 50 \u0026micro;M), and CS\u0026thinsp;+\u0026thinsp;SNP (15\u0026thinsp;+\u0026thinsp;25 and 30\u0026thinsp;+\u0026thinsp;50), under irrigation with four PEG concentrations (0, 5, 10 and 15%) after 1 d. These culture conditions were designed based on preliminary experimental works and literature review \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. After 7 d, the samples were re-sprayed with the same elicitors. The plants undergone the PEG-induced drought for three weeks, and subsequently were harvested in the vegetative growth stage. The harvested plants were washed with dd H\u003csub\u003e2\u003c/sub\u003eO and then used for chemical assays immediately. The leaves were also double washed with dd H\u003csub\u003e2\u003c/sub\u003eO to remove any residual materials on the leaves and then dried in an air-circulated oven at 65\u0026deg;C for 24 h. The fresh weight to dry weight conversion factor was determined 5.79.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eChemical assays\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003ePhotosynthetic pigments\u003c/h2\u003e \u003cp\u003eThe contents of photosynthetic pigments including chlorophyll \u003cem\u003ea\u003c/em\u003e, \u003cem\u003eb\u003c/em\u003e (Chl \u003cem\u003ea\u003c/em\u003e, Chl \u003cem\u003eb\u003c/em\u003e) and carotenoids (C\u003csub\u003ex+c\u003c/sub\u003e) were determined according to the standard method \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. The fresh spinach leaves (0.5 g) were homogenized in acetone 80% and then centrifuged at 15,000\u0026times;g for 30 min. The absorbance of the supernatant was read at the wavelengths of 646.8, 663.2, and 470 nm using a spectrophotometer (UV-Visible 160, Shimadzu, Japan). The contents of pigments were then estimated by the following empirical correlations \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eChl a\u003c/em\u003e (mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;12.25 \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e663.2\u003c/em\u003e\u003c/sub\u003e \u0026ndash; 2.79 \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e646.8\u003c/em\u003e\u003c/sub\u003e (1)\u003c/p\u003e \u003cp\u003e \u003cem\u003eChl b\u003c/em\u003e (mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;21.51 \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e646.8\u003c/em\u003e\u003c/sub\u003e \u0026ndash; 5.10 \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e663.2\u003c/em\u003e\u003c/sub\u003e (2)\u003c/p\u003e \u003cp\u003e \u003cem\u003eC x\u0026thinsp;+\u0026thinsp;c\u003c/em\u003e (mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) = (1000 \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e470\u003c/em\u003e\u003c/sub\u003e \u0026ndash; 1.82 \u003cem\u003eChl a\u003c/em\u003e \u0026ndash; 85.02 \u003cem\u003eChl b\u003c/em\u003e) /198 (3)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHydrogen Peroxide\u003c/h2\u003e \u003cp\u003eThe contents of hydrogen peroxide were measured by the standard method \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The spinach leaves (0.1 g) were ground to homogenization in an ice bath with 5 mL TCA 0.1% w/v. The sample was centrifuged at 12,000 \u0026times;g for 20 min. A 0.5 mL aliquot of the supernatant was mixed with 0.5 mL phosphate buffer (10 mM, pH 7.0) and 1 mL KI (1 M). The solution absorbance was then read at 390 nm using the spectrophotometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMalondialdehyde\u003c/h2\u003e \u003cp\u003eThe contents of MDA, as a biomarker of lipid peroxidation, was measured using the standard method \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The spinach leaves (0.1 g) were homogenized in 5 mL TCA 0.1%w/v. The extract was centrifuged at 8000\u0026times;g for 15 min. The supernatant (1 mL) was mixed with TCA/TBA reagent (4 mL) and the solution was heated at 95\u0026deg;C for 30 min and then cooled on ice immediately. The solution was centrifuged at 8000\u0026times;g for 15 min. The supernatant\u0026rsquo;s absorbance was read 532 nm and the value was subtracted from the non-specific absorbance reading at 600 nm. The MDA contents were estimated using an extinction coefficient of 155 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and expressed as mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFlavonoids\u003c/h2\u003e \u003cp\u003eThe contents of flavonoids were determined by following the standard method in literature \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. In brief, the spinach leaves (1 g) were ground to homogenization in acetic acid-ethanol (1:99 v/v) using a pre-chilled mortar and pestle. The tissue homogenate was centrifuged at 12,000\u0026times;g and 4\u0026deg;C for 15 min. The supernatant was incubated in a water bath (80\u0026deg;C, 10 min) and then cooled down to room temperature. The sample absorbance was read at 270, 300, and 330 nm by the spectrophotometer. The concentration of flavonoids was estimated by the extinction coefficient of 33,000 mol\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e cm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, and expressed as \u0026micro;mol per g dry weight (DW).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAnthocyanins\u003c/h2\u003e \u003cp\u003eThe anthocyanin content was determined according to the standard method \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In brief, the harvested leaves (1 g) were homogenized in HCl-methanol solvent (1:99 v/v) using a pre-chilled mortar and pestle. The homogenate suspension was centrifuged at 12,000\u0026times;g for 10 min. The supernatant was incubated in darkness at 4\u0026deg;C for 24 h, and then its absorbance was read at 550 nm by the spectrophotometer. The anthocyanin\u0026rsquo;s concentration was quantified by the extinction coefficient of 33,000 mol\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e cm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, and expressed as \u0026micro;mol per g leaf DW.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePhenols\u003c/h2\u003e \u003cp\u003eTotal phenol content was determined by the available method in literature \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. In brief, the harvested leaves (1 g) were ground to homogenization in 1 mL dd H\u003csub\u003e2\u003c/sub\u003eO (80%) at 4\u0026deg;C, and centrifuged at 15,000\u0026times;g for 15 min. A 100 \u0026micro;L aliquot of the diluted extract (1:20) was mixed with 50 \u0026micro;L Folin-Ciocalteu reagent (2 N), and the mixture was incubated at 25\u0026deg;C for 5 min. Thereafter, a 20% sodium carbonate solution (300 \u0026micro;L) was added to the reaction and the solution was further incubated at 25\u0026deg;C for 15 min. Ultimately, the sample was centrifuged at 12,000\u0026times;g for 5 min, and the supernatant\u0026rsquo;s absorbance was read at 725 nm by the spectrophotometer. The total phenol was quantified by a calibration curve method using gallic acid as standard and expressed as \u0026micro;mol GAE per g leaf DW.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMinerals\u003c/h2\u003e \u003cp\u003eThe spinach leaves were washed with dd H\u003csub\u003e2\u003c/sub\u003eO and the sample was then dried in an air-circulated oven until reach to the constant weight. The minerals (Ca, Na, K, Fe, Mg, and P) were then extracted from the dried plants (0.5 g) by digestion in a mixture of concentrated nitric acid and perchloric acid (2:1v/v). Subsequently, the solution was analyzed using an inductively coupled plasma optical emission spectrometry (ICP-OES), Model VARIAN 725-ES, America, under optimized operating conditions. The concentrations of Fe, Mg, P, Na, K, and Ca were determined at the specific wavelengths of 261.2 nm, 279.8 nm, 213.6 nm, 589.6 nm, 766.5 nm, and 317.9 nm, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll the cultures and treatments of the spinach plants were done in triplicates. The plant leaves were sampled and used for further chemical assays. The means of the data were then analyzed by one-way analysis of variance in the SPSS Statistics Version 22.0 to identify statistically significant differences at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The results were presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by Iran National Science Foundation, Tehran-Iran, (Grant number 4014890).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eBoth authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Faezeh Khatami. Supervision and data analysis was done by Vahid Niknam. The first draft of the manuscript was written by Faezeh Khatami. Both authors read and approved the final manuscript for submission.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eProvision of the laboratory facilities by University of Tehran is greatly appreciated. We are thankful to the financial support by Iran National Science Foundation (Tehran, Iran) under grant number 4014890. Proof reading and editing by Hamyarapply Group (Tehran, Iran) is greatly appreciated.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKhatami, F. \u0026amp; Niknam, V. 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UV-B response of cucumber seedlings grown nder metal halide and high pressure sodium/deluxe lamps. \u003cem\u003ePhysiol. Plant.\u003c/em\u003e \u003cb\u003e88\u003c/b\u003e, 350\u0026ndash;358 (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWagner, G. J. Content and vacuole/extravacuole distribution of neutral sugars, free amino acids, and anthocyanin in protoplasts. \u003cem\u003ePlant Physiol.\u003c/em\u003e \u003cb\u003e64\u003c/b\u003e, 88\u0026ndash;93 (1979).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDaley, J., Seevers, P. M. \u0026amp; Ludden, P. Studies on wheat stem rust resistance controlled at the Sr6 locus. III. Ethylene and disease reaction. \u003cem\u003ePhytopathology\u003c/em\u003e \u003cb\u003e60\u003c/b\u003e, 1648\u0026ndash;1652 (1970).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Footnotes","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e FAO Stat 2024, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://data.un.org/data.aspx\u003c/span\u003e\u003cspan address=\"https://data.un.org/data.aspx\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"drought, mineral nutrients, phenolics, photosynthesis, Spinacea Oleracea, chitosan, sodium nitroprusside","lastPublishedDoi":"10.21203/rs.3.rs-9328377/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9328377/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDrought perturbs water potential in the plants, led to oxidants accumulation and impaired cellular functions. The mineral nutrients are critical for adjusting water potential and modulating antioxidant activity and photosynthesis. The present study investigated the impact of sodium nitroprusside (SNP) and chitosan (CS) on six key mineral nutrients (Na, K, P, Ca, Mg, Fe) in spinach exposed to polyethylene glycol (PEG)-induced drought. The 3-leaved seedlings were irrigated with PEG (5%, 10%, and 15%) and treated by foliar spray of SNP (25 and 50 \u0026micro;M) and CS (15 and 30 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The physiological responses were studied by measuring the concentrations of hydrogen peroxide, malondialdehyde, chlorophylls, carotenoids, phenols, flavonoids, anthocyanins, and nutrients using UV/Vis spectroscopy and inductively coupled plasma optical emission spectrometry. Increasing drought intensity enhanced hydrogen peroxide accumulation and malondialdehyde. Drought stress led to higher Mg, while Fe remained constant due to its dual function in generating oxidants via Fenton reaction and stimulating antioxidants and photosynthesis. The SNP and CS application enhanced photosynthesis and alleviated the PEG-induced oxidative stress by enhanced production of phenolics and carotenoids. Both elicitors increased Ca, P and Fe contents and decreased Na, K and Mg contents. The intricate interplay between all six nutrients were critical to adjust water potential, minimize oxidative damage, and improve photosynthetic performance. Overall, the cross-talks between Fe, Mg and Ca were important for photosynthetic performance and antioxidant activity. The Na, K and P interplay was essential for osmotic adjustment.\u003c/p\u003e","manuscriptTitle":"Sodium nitroprusside and chitosan alleviate drought stress in spinach by modulating nutrient balance, phenolic production, and photosynthesis ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-21 14:55:19","doi":"10.21203/rs.3.rs-9328377/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-04T18:13:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-27T20:55:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-25T08:01:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223505205611190356243912296639631894207","date":"2026-04-16T08:09:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"92738177154074974976933331762600351224","date":"2026-04-15T10:37:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"145028960918993825159712308712540941497","date":"2026-04-14T07:38:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-14T04:39:14+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-13T18:51:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-07T12:42:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-07T12:41:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-05T20:31:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2db2186e-8b19-4ea8-bdb2-e2a9e5bda6ae","owner":[],"postedDate":"April 21st, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-04T18:13:09+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":66616687,"name":"Biological sciences/Biochemistry"},{"id":66616688,"name":"Biological sciences/Physiology"},{"id":66616689,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-05-04T18:24:10+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-21 14:55:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9328377","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9328377","identity":"rs-9328377","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}