Drought Stress Alleviation by Potassium-Nitrate-Containing Chitosan/Montmorillonite Microparticles Confers Changes in Spinacia oleracea L.

: Drought and low amounts of mineral nutrients in the soil are the two leading global constraints in arid and semiarid regions. Their detrimental effects on soils and crops can be alleviated by applying controlled release and biodegradable fertilizers to better and sustain the crops. On a global scale, spinach ( Spinacia oleracea L.) is an essential leafy green vegetable that is biologically considered a reliable source of essential nutrients and minerals for human health. A comprehensive approach is needed to manage water stress to mitigate the impacts of stress-caused damage and to examine this for better and increased plant production. An experiment was conducted using potassium-nitrate-containing chitosan/montmorillonite microparticles (150 mg) under mild and severe drought stress (MDS: 50% and SDS: 35% FC, respectively). The treatments include control (no KNO 3 and 70% FC as normal irrigation (NI)), KNO 3 + NI, 50% FC as mild drought stress (MDS), KNO 3 + MDS, 35% FC as severe drought stress (SDS) and KNO 3 + SDS. Results revealed that drought stress decreased all studied physiological parameters and increased oxidative stress indicators in spinach. Applying KN signiﬁcantly increased root (122%) and shoot length (4%), shoot fresh weight (32%) and shoot dry weight (71%), chlorophyll a (88%), carotenoids (39%), total soluble proteins (50%), soluble sugars (51%), potassium (80%), and phosphorous (32%) concentrations over No KN at severe drought. While stress indicators, like glycine betaine, malondialdehyde, hydrogen peroxide, electrolyte leakage, peroxidase, superoxide dismutase, and ascorbic acid levels, were increased in stress. Treatment KN was proved efﬁcient and effective in improving spinach physiological status in both MDS and SDS.

So far, many scientists have documented the positive role of K against drought stress. Yet, limited work is documented on the use of potassium nitrate containing chitosan/montmorillonite microparticles. Therefore, the present study was conducted to check the potential effects of potassium nitrate containing chitosan/montmorillonite microparticles on spinach under drought stress. It is hypothesized that potassium nitrate (containing chitosan/montmorillonite microparticles) might be effective for better spinach growth under drought stress.

Experimental Site, Design, and Treatment Plan
A pot experiment (2019) was performed at Old Botanical Garden, University of Agriculture, Faisalabad. Geographically, Faisalabad is located at 31.4504 • N, 73.1350 • E at 610 ft above the mean sea level in flat plains of northeast Punjab in a semi-arid temperature zone. Soil (15-20 cm depth) was obtained from university research site and then dried and sieved through a 2-mm sieve. Then, the soil was uniformly packed in pots (25-cm diameter × 18-cm height). Some important soil characteristics include electrical conductivity (EC), 7.82 (dS/m); organic matter (OM), 0.68%, pH, 8.1; total nitrogen (N), 0.034%; available phosphorus (P), 6.13 mg/kg; and extractable potassium (K), 123 mg/kg. The experimental design was a completely randomized block design (CRD) and replicated four times. The treatments include control (no potassium nitrate under 70% FC as normal irrigation (NI)); KNO 3 + NI, 50% FC irrigation as mild drought stress (MDS); and KNO 3 + MDS, 35% FC irrigation as severe drought stress (SDS), KNO 3 + SDS.

Fertilizer Preparation
Mechano-chemical intercalation of KNO 3 and montmorillonite (MM) was done using agate mortar by grinding 75% by weight of KNO 3 in distilled water and montmorillonite in 1:3 w/w of MM/KNO 3 . Then, MM/KNO 3 was dried in the oven and crushed into powder. Meanwhile, 4 g chitosan (C) mixed in 100 mL of acetic acid solution (2% v/v) was kept at 25 • C for a day. C/MM-KNO 3 was made by mixing 16 g MM-KNO 3 with chitosan solution to have final ratio of 1:4 of C/MMt-KNO 3 . Then, using a mechanical stirrer, the homogenous dispersions were maintained at 12.000 rpm for 1 h. Complete chitosan crosslinking was achieved at two different volumes of sodium tripolyphosphate solution (32 and 64 mL of 1% and 5% w/v, respectively) to C/MM-KNO 3 dispersions. After this, the solution was dried using a spray dryer (1.5-mm nozzle cap) to ensure a completely dry material [21]. There were 2 levels of potassium nitrate (KNO 3 ), i.e., control (having no KNO 3 ) and 150 mg KNO 3 pot −1 . Potassium-nitrate-containing chitosan/montmorillonite microparticles were added to soil at the concentration of 150 mg pot −1 at the time of sowing.

Plant Material and Seed Sowing
Seeds of Spinach were collected from Ayub Agriculture Research Institute, Faisalabad. The seeds were disinfected using ethanol (95%) for 60 s and then with sodium hypochlorite solution (NaOCl; 70%) for 10 min. All seeds were later washed six times with deionized water.

Irrigation and Drought Stress
For irrigation purposes, tap water was used. There were three water regimes, i.e., controlled, normal irrigation (70% FC), mild drought stress (50% FC), and severe drought stress (35% FC). All the irrigations levels were maintained in w/w basis. For determination Sustainability 2021, 13, 9903 4 of 15 of field capacity (FC), 5 kg of soil was taken in a pot. After that, soil was fully saturated with water. The volume of water that was used for saturation of soil was also weighed on analytical grade balance (y). Finally, the pot was left for 24 h so that gravitational water may leached down. The next day, the weight of leachate water (z) was determined on analytical grade balance. The difference in weight provides the amount of water required for achievement of 100%FC of 5 kg soil. From this, 70 and 50% FC for selected soil was calculated by using unit method equations, i.e., For 100% FC weight of water required = (y − z) = x (g) For 1% FC weight of water required = x/100 (g) For 70% FC weight of water required = (x/100) × 70 (g) For 50% FC weight of water required = (x/100) × 50 (g)

Harvesting and Data Collection
The harvest was taken after 25 days of sowing. After harvest, all plants were rinsed twice with distilled water. Fresh weights were measured immediately after harvesting using a digital weighing balance at the experimental site. Plant shoot and root lengths were measured using a measuring tape. Fresh plants samples were stored in biomedical freezer at −30 • C. Samples from each treatment were oven dried for 72 h at 65 • C and were used to measure dry weight and ion analysis.

Determination of Chlorophyll Contents
Chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid contents were assessed following the standard Arnon protocol [22]. A total of 0.1 g of fresh leaf samples were extracted in 95% of acetone (8 mL) at 4 • C temperature in the dark for 24 h. Then, 646-, 663-, and 450-nm absorbance were recorded by spectrophotometer (xMark Microplate Absorbance Spectrophotometer; Bio-Rad, Hercules, CA, USA).

Malondialdehyde (MDA)
MDA contents were evaluated to determine lipid peroxidation extent during oxidative stress. A total of 0.1 g fresh leaf samples were homogenized in 25 mL of 50 mM concentrated phosphate buffer (pH 7.8) having 1% polyethene pyrrole at 4 • C and centrifuged for 15 min at 10,000× g. This mixture was boiled at 100 • C for 20 min and cooled immediately in an ice bath. The absorbance of supernatant liquid in solution was recorded by spectrophotometer (xMark Microplate Absorbance Spectrophotometer; Bio-Rad, Hercules, CA, USA) at 532-, 600-, and 450-nm wavelengths. Peroxidation of lipid components was recorded using protocol by Heath and Packer [23].

Hydrogen Peroxide (H 2 O 2 )
H 2 O 2 estimation of leaf and root samples was done using 3 mL of sample extracts in 1 mL of 0.1% titanium sulfate and 20% H 2 SO 4 (v/v) and then centrifuged for 15 min at 6000× g. The color intensity of samples was analyzed by spectrophotometer at a wavelength of 410 nm following the Jana and Choudhuri method [24].

Electrolyte Leakage (EL)
EL caused by drought stress of flag leaves was estimated following the Dionisio-Sese and Tobita method [25]. The samples were immersed in 8 mL of distilled water in test tubes and incubated for 2 h, and EC 1 (initial electrical conductivity) was measured. The samples extracts were then autoclaved for 20 min at 121 • C temperature and cooled at room temperature, and EC 2 (final electrical conductivity) wula: EL (%) = (EC 1/EC 2) × 100

Antioxidant Enzymes
To assess antioxidant activities, 0.5 g fresh leaf samples were homogenized in liquid nitrogen, 0.15 mol NaCl, 50 mmol of sodium phosphate buffer (5 mL, pH 7.0), and 0.5 mmol EDTA solution. This extracted mixture was centrifuged for 10 min at 12,000× g with 4 • C temperature. The supernatant liquid was used for the evaluation of peroxidase and superoxidase dismutase activities in plants. SOD activity was determined by taking 3 mL sample mixtas measured by the following formure having 50 mM concentrated sodium phosphate buffer of pH 7, 56 mM of nitro blue tetrazolium chloride, 1.17 mM of riboflavin, 10 mM methionine, and 100 mL of enzyme extract. The light intensity of the final reaction mixture was estimated by spectrophotometer, following the method of Chen and Pan [26]. Peroxidase (POD) enzyme activity in the spinach leaf sample was analyzed by the Sakharov and Ardila [27] method. For this, guaiacol substrate was used. A 3-mL mixture was prepared, containing 0.1 mL of 4% guaiacol solution, 0.05 mL of enzyme extract, 2.75 mL of phosphate buffer (50 mM, pH 7.0), and 0.1 mL of 1% H 2 O 2 . Color intensity was recorded at 470 nm wavelength.

Soluble Sugars and Non-Enzymatic Antioxidants
To determine the concentrations of osmolytes and non-enzymatic antioxidant constituents, ethanol extracts of spinach plant samples were prepared using 50 mg of dried plant matter that was extracted in 10 mL of 80% ethanol and filtered using a filter paper and re-extracted in 10 mL ethanol. The 20-mL final volume of the solution was maintained by mixing the two extracts. This reaction mixture was used to determine concentrations of the following: total soluble proteins [28], glycine betaine [29], total sugars [30], and ascorbic acid [31] contents in plants.

Nutrient Concentration
Shoots and roots were washed in redistilled water twice for plant nutrient analysis. The plant samples were oven dried for 72 h at 65 • C. The samples were digested in HNO 3 : HClO 4 (7:3 v/v) using wet digestion protocol until colorless samples were obtained. The samples were filtered, followed by dilution in redistilled water. Volume was maintained up to 50 mL. Yoshida et al. [32] proposed the method for determining calcium, sodium, and potassium ions concentration in plants. Their concentrations were estimated by using Atomic Absorption Spectrum using a flame photometer. Concentrations of unknown samples of respective elements were found by constructing standard curves using standard series. The yellow color method was used to determine phosphorous contents at 420 nm absorbance using spectrophotometer [33].

Statistical Analysis
The data were analyzed with two-way ANOVA (ANOVA), while difference in treatments was computed by least significant difference (p < 0.05) test [34]. For data normalization before analysis, logarithmic transformations were performed where necessary. Pearson's correlation analysis was carried out using Origin 2021.

Root Length, Shoot Length, Shoot Fresh Weight, and Shoot Dry Weight
Application of potassium nitrate (KN) significantly affected the root length, shoot length, shoot fresh weight, and shoot dry weight of spinach under normal irrigation (NI), mild drought stress (MDS), and severe drought stress (SDS). Increasing drought stress caused a significant reduction in root and shoot length, fresh weight, and dry weight in spinach. In NI, treatment KN plants showed significant enhancement in root length ( Figure 1A), shoot length ( Figure 1B), and shoot fresh weight ( Figure 1C). No significant change was observed in shoot dry weight ( Figure 1D) among KN and no KN under NI. Under MDS, application of KN remained significantly better over no KN for root and shoot length, shoot fresh, and shoot dry weight of spinach. However, at SDS, KN differed significantly over no KN for root length, shoot fresh, and dry weights of spinach. Treatment KN and no KN were non-significant for shoot length at SDS. spinach. In NI, treatment KN plants showed significant enhancement in root length (Figure 1A), shoot length ( Figure 1B), and shoot fresh weight ( Figure 1C). No significant change was observed in shoot dry weight ( Figure 1D) among KN and no KN under NI. Under MDS, application of KN remained significantly better over no KN for root and shoot length, shoot fresh, and shoot dry weight of spinach. However, at SDS, KN differed significantly over no KN for root length, shoot fresh, and dry weights of spinach. Treatment KN and no KN were non-significant for shoot length at SDS.

Chlorophyll and Carotenoid Contents
Treatments KN differed significantly for chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids of spinach under NI, MDS, and SDS. Drought stress enhancement significantly decreased all of the photosynthetic pigments of spinach. For NI, addition of KN did not show a significant increase from no KN in chlorophyll a ( Figure 2A) and total chlorophyll ( Figure 1C). However, KN application remained significantly better for chlorophyll b ( Figure 2B) and carotenoids ( Figure 2D

Chlorophyll and Carotenoid Contents
Treatments KN differed significantly for chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids of spinach under NI, MDS, and SDS. Drought stress enhancement significantly decreased all of the photosynthetic pigments of spinach. For NI, addition of KN did not show a significant increase from no KN in chlorophyll a ( Figure 2A) and total chlorophyll ( Figure 1C). However, KN application remained significantly better for chlorophyll b ( Figure 2B) and carotenoids ( Figure 2D) of spinach leaves ( Figure 2B)

K, Na, Ca, and P Concentrations
Effect of KN application was significant for K, Na, Ca, and P of spinach under NI, MDS, and SDS. Results showed that KN showed significant enhancement in K ( Figure 4A) and P ( Figure 4D) but decreased Na ( Figure 4B) and Ca ( Figure 4C) compared to no KN in NI. Under MDS, KN differed significantly for K and P enhancement, while Ca decreased over no KN. However, no significant change in Na was noted at MDS among KN and no KN. Treatment KN was significant for increased K and P but a decrease in Ca and Na of spinach under SDS.

K, Na, Ca, and P Concentrations
Effect of KN application was significant for K, Na, Ca, and P of spinach under NI, MDS, and SDS. Results showed that KN showed significant enhancement in K ( Figure 4A) and P ( Figure 4D) but decreased Na ( Figure 4B) and Ca ( Figure 4C) compared to no KN in NI. Under MDS, KN differed significantly for K and P enhancement, while Ca decreased over no KN. However, no significant change in Na was noted at MDS among KN and no KN. Treatment KN was significant for increased K and P but a decrease in Ca and Na of spinach under SDS.

TSP, SS, and AsA
Application of KN was significantly different for TSP, SS, and AsA of spinach under NI, MDS, and SDS. The addition of KN significantly increased TSP ( Figure 5A) and SS ( Figure 5B) but remained non-significant for AsA ( Figure 5C)

Pearson Correlation and Principal Component Analysis
Pearson correlation showed that the studied growth attributes, i.e., plant length, fresh and dry weights, and photosynthetic pigment contents, were significantly negative in correlation with MDA, proline, POD, SOD, Na, Ca, H2O2, GB, and EL. However, plant length, shoot fresh and dry weight, chlorophyll a, chlorophyll b, and total chlorophyll contents were significant positive in correlation with K, P, TSP, and SS ( Figure 6). The principal component analysis also showed that K, P, fresh and dry weights of shoot, shoot and root length, TSP, and chlorophyll contents were closely associated with each other. However, AsA, GB, POD, SOD, Ca, Na, H2O2, electrolyte leakage, and proline were opposite and far apart from K, P, vegetative growth characters, TSP, and chlorophyll contents. Catalase was more closely associated with K, P, shoot fresh and dry weight, plant height, TSP, and chlorophyll contents than AsA, GB, POD, SOD, Ca, Na, H2O2 electrolyte leakage, and proline (Figure 7).

Pearson Correlation and Principal Component Analysis
Pearson correlation showed that the studied growth attributes, i.e., plant length, fresh and dry weights, and photosynthetic pigment contents, were significantly negative in correlation with MDA, proline, POD, SOD, Na, Ca, H 2 O 2 , GB, and EL. However, plant length, shoot fresh and dry weight, chlorophyll a, chlorophyll b, and total chlorophyll contents were significant positive in correlation with K, P, TSP, and SS ( Figure 6). The principal component analysis also showed that K, P, fresh and dry weights of shoot, shoot and root length, TSP, and chlorophyll contents were closely associated with each other. However, AsA, GB, POD, SOD, Ca, Na, H 2 O 2 , electrolyte leakage, and proline were opposite and far apart from K, P, vegetative growth characters, TSP, and chlorophyll contents. Catalase was more closely associated with K, P, shoot fresh and dry weight, plant height, TSP, and chlorophyll contents than AsA, GB, POD, SOD, Ca, Na, H 2 O 2 electrolyte leakage, and proline ( Figure 7). Sustainability 2021, 13, x FOR PEER REVIEW 11 of 15

Discussion
Spinach is a valuable leafy green vegetable and a good source of nutrients. Like most vegetables, limited water availability badly affects spinach production in semi-arid areas that require proper irrigation systems, but water scarcity is a significant limiting factor. Moreover, the extensive use of synthetic chemical fertilizers has caused great damage to agricultural system. The use of controlled-release fertilizers that are biodegradable to increase yield has recently gained significant momentum for agricultural sustainability [35]. Drought disturbs water relations and impairs normal plant growth. Plants, however, have many biochemical response mechanisms at the cellular and whole-organism level, making drought stress an intricate phenomenon [36].
According to Gargallo-Garriga et al. [37], limited water availability due to drought stress restricted shoot metabolic activities. Lower metabolic rates facilitate plants to conserve water and transfer assimilates from upper parts towards the root. This maintenance of water status and food transfer helps plants to make extensive root systems and absorb more water from the soil. Our study also validates these facts. Elongated roots were observed in spinach plants treated with KN in both water deficit conditions, MDS, and SDS. Extensive cell division is the estimation of improved plant growth. Drought stress changes soil osmotic potential, leading to poor growth and cell division in roots and shoot. Less water uptake due to change in the osmotic potential decreases the fresh weight of root and shoot. It also results in less division and a significant reduction of dry weight [38,39]. Similar results were noticed in the current study. KN treatment effectively increased shoot fresh weight and plant length by conserving more water contents in cells.
Chlorophyll b, a premier light-harvesting pigment, experiences more reduction than chlorophyll a because water deficiency mainly affects photosystem-I, light harvesting systems, antenna complex, and energy-transfer mechanisms. Drought stress significantly reduces chlorophyll b contents in spinach plants even at 80% field capacity [20]. Our results are also in justification of these mechanisms. A significant decrease in photosynthetic pigment contents of spinach was observed under MDS and SDS conditions [40,41]. Application of potassium fertilizer at 150 mg concentrations significantly enhanced chlorophyll b levels. KN also improved plant vigor and photosynthetic activities in drought stress, from which chlorophyll a, total chlorophyll, and carotenoids were also improved.
Cellular osmotic adjustment is important to cope with water stress conditions. Plants maintain turgor pressure in cells and regulate several physiological processes to postpone and tolerate the prevailing dehydration conditions. Drought stress denatures protein and enzyme structure, and to protect them, plants use osmo-protectants [42]. Higher sugar levels are maintained in cells to protect denaturalization of proteins in water stress. Similar results were reported by Ahmed et al. [42] and Kishor et al. [43]. Osmoregulation maintains turgor potential in cells and improves physiological processes to cope with prevailing dehydration [6].
ROS cause peroxidation of membranous lipids, and the extent of damage is assessed by malondialdehyde, a lipid peroxidation product. The spinach plant's amount of malondialdehyde (MDA), ascorbic acid, glycine betaine, and proline increases under water stress conditions. Levels of hydrogen peroxide (H 2 O 2 ) and antioxidant enzymes (SOD, POD, CAT) remained unaffected even at the levels of 40% field capacity in spinach [20]. The higher synthesis of MDA, phenolics, and H 2 O 2 in the present study also validated the mitigation of severe water stress under KN. H 2 O 2 has various physiological and growth-related roles in plants, thus ameliorating drought resistance in spinach.
According to Coskun et al. [44], MDA contents positively correlated with Na + ion uptake in stressed plants. Controlled amounts of Na + ions are very critical to stress tolerance in plants. Limited amounts of ions prevent ion imbalances, leaf metabolic disorders, and tissue desiccation via osmotic stress. To maintain proper cellular functions, intracellular K + ions must be maintained in homeostatic concentrations. Plants with efficient absorption and utilization of mineral nutrients effectively increase fertilizer capability to affect plant vigor, thus reducing production cost and nutrient loss to the ecosystem. Plants with high nutrient-use efficiency contribute to a sustainable agriculture system and better water, soil, and air qualities [45]. Our results clearly state that KNO 3 fertilization increased Na + , Ca 2+ , and K + ion contents and their uptake in spinach. Na + was relatively in greater amount in plants under drought stress.

Conclusions
In conclusion, the application of KNO 3 containing chitosan/montmorillonite microparticles under drought positively affects spinach's growth, physiological, and ionic attributes. Plant physiological parameters, like length, fresh weight, dry weight, chlorophyll pigments, carotenoids, total soluble protein contents, soluble sugars, potassium, and phosphorous, were significantly improved with the supplementation of KNO 3 in mitigating the effects of drought stress. KN had efficacious roles in alleviating drought stress in spinach plants.
There is a need for more investigation at the field levels considering other climatic conditions to validate these results using KNO 3 containing chitosan/montmorillonite fertilizer as an effective amendment against drought.