Effect of acetylsalicylic acid and ammonium sulphate on productive and physiological parameters in Stipa caudata under water shortage conditions

Stipa caudata is a grass native to low rainfall areas in Argentina and Chile, considered an excellent potential candidate for biofuel production or soil restoration programmes. This study aimed at analysing the effects of ammonium sulphate (AMS) and acetylsalicylic acid (ASA) on the productivity and biochemical traits of plants of this species under water scarcity conditions. The experimental work was carried out on plants grown outdoors using a randomised block plot design. Several yield and biochemical parameters related to resistance to water scarcity were analysed in plants treated with AMS or ASA. Plants in the treatments with ASA and AMS had higher total chlorophyll content than the others. Concerning ion content, water-restricted plants treated with AMS had similar values to irrigated plants. Regarding the osmoprotectants and antioxidants, treated plants had increased concentrations of proline and total flavonoids. Under water stress, plants had higher APX activity and there was an A x B interaction for CAT and SOD activity. The results obtained show that the use of ASA and AMS in some crops or in environmental restoration programmes could be a useful tool to cope with future climate scenarios of water scarcity. Pro was expressed as mmol g DW.


Introduction
Chile's latest climate change projections predict that there will be an increase in temperatures and a decrease in rainfall in much of the north and centre of the country, leading to desertification and reduced water 2 availability for plants (IPCC, 2000;Masson-Delmotte, 2018). Water stress is one of the leading environmental problems affecting crops. It is associated with several physiological and biochemical cellular effects, including oxidative damage by increased reactive oxygen species (ROS) production, reducing plant growth and productivity (Osakabe et al., 2014;Sun et al., 2020).
Stipa caudata Trin. is a grass native to Chile and Argentina, adapted to areas of low annual rainfall (100 -300 mm/year) and agriculturally infertile and eroded soils. The species is a good candidate for soil restoration and biofuel production programmes. Plants tolerant to water stress can respond through a variety of physiological changes, such as an increase of osmoprotectant levels or the activation of antioxidant systems. Under stress conditions, plants increase the synthesis of osmolytes, such as proline, an amino acid involved in responses to various stresses (Magdy et al., 2017;Vargas-Ortiz et al., 2021). Antioxidant mechanisms are based on the synthesis of metabolites with strong antioxidant activity, such as phenolic compounds and especially the subgroup of flavonoids, and the activation of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) or glutathione reductase (GR), which help to maintain the plant's metabolic functions, minimising inhibition of growth (Apel and Hirt, 2004).
To mitigate environmental constraints in dry areas, the use of various additives has shown promising results. For example, nitrogen and sulphur are essential mineral elements for plants, partly because they participate in the synthesis of glutathione, a tripeptide involved in the detoxification of ROS (Neuberg et al., 2010). Moreover, since nitrogen assimilation is crucial for ion uptake and amino acids and proteins synthesis, ammonium sulphate is one of the most important nitrogen fertilisers. On the other hand, acetylsalicylic acid (ASA) is a precursor of salicylic acid, a phytohormone that plays an essential role in plant signalling in response to various stresses, including water stress (Nazar et al., 2015).
However, there is very little information on the physiological responses for increased resistance and biomass production under water deficit conditions in Stipa caudata. Even less is known on the possible effects of sustainable strategies such as ammonium sulphate (AMS) or acetylsalicylic acid (ASA) application to waterstressed plants. This study represents a first assessment of the resilience of Stipa caudata to water stress and on the alleviation of this stress by sustainable and low-cost strategies.
Therefore, this work aimed at evaluating the effects of the application of AMS and ASA on biomass production and activation of specific stress tolerance mechanisms of Stipa caudata plants under water deficit conditions by (i) analysis of growth parameters in control and stressed plants; (ii) quantification of leaf contents of total chlorophyll, ions and osmolytes; (iii) determination of total phenolic compounds and flavonoids concentrations in shoots; (iv) measurement of the specific activities of several antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase).

Study site and experimental conditions
The experiment was conducted under field conditions in September 2018, at the Bosque Santiago facilities, Ministry of Housing and Urbanism, Government of Chile, located at Camino La Pirámide 6.000, Huechuraba, Metropolitan Region, Chile (33° 22′ 38.6″ S 70° 36′ 3.36.8″ W). The temperature range was between 28 to 31.6 °C, with an average of 22 °C and a mean relative humidity of 67.2%, without precipitation (Agromet, 2018).
Analyses were carried out in the Faculty of Sciences of the Universidad Mayor, Santiago, Chile and COMAV, Polytechnic University of Valencia, Spain.
Before starting the experiment, the plants were conditioned for six weeks in the open, watering them daily up to field capacity (FC) with irrigation water from the Bosque Santiago facility. Basal fertilisation was performed 23 days after transplanting with a nutrient solution based on 0.1% total N and 3% K at a dose of 1.5 L/ha. The same dose was repeated on day 37 after transplanting. The plants were pruned twice and cut at the height of 10 cm from the ground; the first pruning was performed on the same day as the first basal fertilisation and the second on the day starting the treatments.
The experimental design used was a randomised block plot design (RBLP) with four replicates per treatment. The main plot corresponded to the water status (with or without irrigation), and the sub-plot corresponded to the ammonium sulphate and acetylsalicylic acid treatments.
Plants were treated at the beginning of the experiment in 10 L plastic pots containing 19% clay, 26% lime and 55% sand andisol soil (pH = 7,2, EC = 0,47 dS/m, Cox = 1,5 %) with 0.5 mM ASA (0.09 g/plant) diluted in 30 mL of distilled water and 2 g per pot AMS in irrigated and non-irrigated groups. In the treatment with irrigation, plants were watered every two days until reaching the pot weight corresponding to FC. In the water stress treatment, the stress period consisted of withholding irrigation for 14 days, when ca. 90% of the non-irrigated plants showed symptoms of severe general chlorosis. Each plant of Stipa caudata in a pot represented one experimental unit (n = 32). Table 1 indicates the experimental design consisting of four randomised blocks, each with two variants: with and without irrigation.  WS   T1  T3  T4  T2  T3  T3  T1   T2   T4  T4  T1  T1  T2  T1  T4   T4   T2  T2  T2  T4  T1  T2  T2   T1   T3  T1  T3  T3  T4  T4  T3 T3 T1 = ASA + AMS; T2 = ASA; T3 = AMS; T4 = Control.

Evaluation of plant morphological and biochemical traits
The length of five vegetative shoots per plant was determined using a ruler. The number of spikes was also registered every week. At the end of the experiment, plants were harvested, part of the aerial biomass was frozen, and the rest was dried in an oven at 65 °C to a constant weight.
Total chlorophyll content was measured according to a previously described method (Walinga et al., 1989). Stem material (0.1 g fresh weight) was crushed in a mortar in the presence of liquid N2. For extraction, 1 ml of 80% cold acetone was used. Samples were shaken at 4 °C overnight and then centrifuged at 12,000 rpm for 10 min. The supernatant was diluted 10-fold in 80% acetone. A spectrophotometer (VWR UV-1600PC) was used for the measurements.
Ion (Na + , Cl -, K + and Ca 2+ ) contents were determined according to Weimberg (1987), in leaf water extracts obtained by heating ground, dry plant material (0.15 g in 10 mL of water) in a water bath for 1 h at 95 °C. Cations were analysed with a flame photometer (Corning 410 C), and Clwas measured with a chloride analyser (Corning 926).
Proline (Pro) content was determined at the end of the experimental period in 0.2 g fresh plant material by the ninhydrin-acetic acid method described by Bates (1973). Briefly, Pro was extracted in 3% aqueous sulphosalicylic acid, and the extract was mixed with acid ninhydrin solution, incubated for 1 h at 95 °C, cooled on ice and then extracted with two volumes of toluene. The absorbance of the organic phase was measured at 520 nm, using toluene as a blank. Pro concentration was expressed as mmol g -1 DW.

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The MDA content was analysed in 0.1 g of fresh leaf material, ground to a fine powder in a mortar and extracted with 1.5 ml of 80% methanol. The samples were shaken gently overnight at 7 °C. The supernatant was collected by centrifugation at 13,300 g for 10 min at 4 °C and stored at -20 °C until used in the assays.
MDA in the extracts was quantified by the trichloroacetic/thiobarbituric acid method as described previously (Hodges et al., 1999).
Total phenolic compounds (TPC) and total flavonoids (TF) were measured in the same methanol extracts used for MDA determination. TPCs were quantified by measuring absorbance at 765 nm after reaction with the Folin-Ciocalteu reagent (Blainski et al., 2013), and expressed as gallic acid equivalents (mg eq GA / g DW), used to obtain the standard curve. TF were measured following the procedure described by Zhisen et al. (1999), based on the nitration of aromatic rings bearing a catechol group and their reaction with AlCl3 under alkaline conditions. After the reaction, the absorbance of the sample was measured at 510 nm, and flavonoids contents were expressed as catechin equivalents (mg eq. Catec. g -1 DW).

Activity of the antioxidant enzymes
Crude protein extracts were prepared from the leaf material, stored frozen at -75 °C, as described by Gil et al. (2014). The protein concentration in the extracts was determined according to Bradford (1976) using the Bio-Rad reagent and bovine serum albumin (BSA) as the standard.
Superoxide dismutase (SOD) activity was determined according to a published protocol (Beyer and Fridovich, 1987) by spectrophotometric monitoring of nitroblue tetrazolium (NBT) photoreduction inhibition. The reaction mixtures (1 ml) contained 50 mM potassium phosphate buffer, pH 7.8, 9.9 mM Lmethionine, 58 mM NBT, 0.025% (v/v) Triton X-100, 2.4 mM riboflavin (as a source of superoxide radicals) and the protein extract. After adding riboflavin, the reaction mixtures were irradiated (300 mmol m -2 s -1 , provided by three compact fluorescent lamps (Osram DULUX PRO 23 W) for 10 min at 25 °C, and the absorbance was measured at 560 nm using a non-irradiated solution mixture as blank. One SOD unit was defined as the amount of enzyme causing 50% inhibition of NBT photoreduction under the assay conditions. Catalase (CAT) activity was determined following the decrease in absorbance at 240 nm that accompanied H2O2 consumption after adding protein extracts to a solution of 10 mM H2O2 in 50 mM Tris-HCl (pH 7.0). A CAT unit was defined as the amount of enzyme that decompose one mmol of H2O2 per minute at 25 °C (Aebi, 1984).
Ascorbate peroxidase (APX) activity was determined by measuring the decrease in absorbance at 290 nm as ascorbate is oxidised in the reaction (Nakano and Asada, 1981). One APX unit was defined as the amount of enzyme required to consume one mmol of ascorbate per minute at 25 °C.
Glutathione reductase (GR) activity was determined according to Connell and Mullet (1986) following the oxidation of NADPH, the cofactor in the GR-catalysed reduction of oxidised glutathione (GSSG). In a final volume of 1 ml, the reaction mixtures contained 100 mM Hepes, pH 7.5, 1 mM EDTA, 3 mM MgCl2, 0.5 mM GSSG and the protein extracts. The reactions were initiated by adding NADPH to a final concentration of 0.2 mM. Samples were incubated at 25 °C, and the decrease in absorbance at 340 nm was measured after 25 min. Control reactions without protein extract were incubated in parallel to correct for nonenzymatic oxidation of NADPH. One GR unit was defined as the amount of enzyme that will oxidise one mmol of NADPH per minute at 25°C.

Statistical analysis
The experiments results were analysed using linear and mixed models to assess whether there were significant differences between treatments, and a Fisher's test for comparison of means, with 95% confidence, to determine differences between treatments. The temporal correlation for the variables shoots growth, the number of spikes, pH and EC were considered in the model when running the analyses. The statistical analysis was conducted using InfoStat, version 2019 (University of Cordoba, Argentina).

Vegetative variables
Lengths of stems were checked every week during the stress treatment. Figure 1 shows the interaction between Treatments AMS/ASA (A) x Water condition (B) for the stem length increment at the end of the experiment. Plants treated with the irrigated AMS and ASA mixture showed the greatest increase in length. In turn, plants with AMS, ASA or the mixture of both, presented the highest levels of stem length regardless of the hydric condition in which they were found. Finally, control plants without treatments and without irrigation showed the lowest values for stem length increase.  Chlorophyll content Total chlorophyll content measured in shoots of S. caudata in the four treatments is indicated in Figure   3. As for the previous trait analysed, the absence of irrigation did not significantly decrease the content of total chlorophyll, but the effect of the treatment was significant. Plants grown on soils fertilised with the mixture of AMS and ASA had significantly higher values of total chlorophyll concentrations than control plants or those from the treatments with AMS and ASA separately.

Ion contents of shoots
Monovalent and divalent cations (Na + , K + , and Ca 2+ ) and the monovalent anion Clwere quantified in the shoots of plants at the end of the experiment. Generally, the effect of treatments and water conditions were not significant.
The only significant interaction between the type of treatment (A) and the water condition (B) was found for the shoots Ca 2+ content, as shown in Figure 4.

Biochemical variables
Several biochemical characteristics, which generally are considered as suitable markers of stress, were measured in shoots of all plants at the end of the treatments and their concentrations are indicated in Table 3.
Variation of the concentration of proline was dependent only on the water regime, but not on the treatments applied. Water stress induced a significant increase in proline concentrations in plants under all treatments (Figure 5a), but interaction between Treatment (A) x Water Stress (B) had no statistical significance. Malondialdehyde (MDA) increased in all watered-stressed plants (Table 3 and Figure 5b) but as for proline neither treatment or interactions between treatments and water conditions were significant. 8 Table 3. Concentrations of proline (Pro), malondialdehyde (MDA), total phenolic compounds (TFC) and total flavonoids (TF) in shoots of Stipa caudata at the end of the experiment.

Water condition (A)
Treatments (B) TFC (mg eq. GA g -1 FW) TF (mg eq.catequin g -1 FW)  Regarding the non-enzymatic antioxidants, the pattern of variation according to the water regime was similar for total phenolics (TFC) and total flavonoids (TF), with an increase in water-stressed plants from all treatments. In the case of TFC, there were detected also significant differences according to the different treatments and the interaction between treatments and water regime. Figure 6 shows the interaction of Treatments (A) x Water condition (B) for TFC content in shoots measured at the end of the experiment. The total of plants treated with ASA, AMS or the mixture of both in the water restricted condition, together with plants treated with ASA with irrigation, presented higher levels of TFC than plants treated with the ASA-AMS mixture with irrigation.  There were significant differences with respect to the water condition (B), where plants under water stress condition (WS) presented higher levels of TF than irrigated plants. Antioxidant enzyme activity Table 4 summarises the activity of Catalase (CAT), Ascorbate Peroxidase (APX), Superoxide Dismutase (SOD) and Glutathione Reductase (GR) in shoots of Stipa caudata measured at the end of the experiment. There was an interaction between treatments (A) x water condition (B) for CAT and SOD activity, together with significant differences for water condition (B) with respect to APX activity. As can be seen in Figure 8, there is an interaction between the hormone/mineral x water condition treatments. There were no significant differences in plants with the hormone/mineral mixture irrigated and not irrigated with respect to the rest of the treatments. There were also no significant differences between control treatments and the rest, regardless of the water condition of the plants. Irrigated plants from the treatments with ASA showed the lowest SOD activity levels, significantly different of all other values calculated, whereas the highest were determined in irrigated and water stressed plants from control treatments.

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ASA irrigated plants showed also the lowest levels of CAT activity compared to the other treatments. On the other hand, irrigated plants from control had the highest CAT activity (Figure 9). Figure 10 shows the APX activity measured in Stipa caudata shoots at the end of the experiment.
Significant differences were found with respect to the water condition (B), where water-restricted plants (WS) showed higher levels of APX than irrigated plants but differences between treatments or interactions water regime x treatments were both not significant.

Productive and physiological variables
The above results show interaction AxB in the studied factors. It was observed that plants from AMS and ASA under water restriction regime produced similar stem height as the irrigated ones. In turn, AMS plants showed the highest chlorophyll contents.
Positive results on height and biomass of water stressed plants treated with ASA or AMS have been reported in tomato (Rodriguez et al., 2020), bean (Seranatna et al., 2000), wheat (Kareem et al., 2017), chickpea (Hussain et al., 2020) and Eragrostis plana (Bastiani et al., 2021). However, several studies (Kabiri and Naghizadeh, 2015) found no effect in height of barley. On the other hand, water-restricted plants had higher MDA contents than irrigated plants. In turn, plants with AMS and ASA showed higher total chlorophyll content than the rest of the treatments. Similar observations have been reported with AMS or ASA treatments in maize (Gunes et al., 2007), Allium sativum (Zeinali and Moradi, 2015), mustard (Giansoldati et al., 2012) and chickpea (Hussain et al., 2020).
The above results suggest that applications of ASA or AMS could increase some growth parameters in water-stressed plants, as they may have an effect on stomatal closure and maintenance of water flow related to growth (Vargas-Ortiz et al., 2021). It is likely that low-dose applications of these supplements at the onset of a water deficit could also have a synergistic effect on stress defence systems. In this sense, Jopia et al. (2020) showed that water deficit could decrease growth by reducing the water potential. Stomatal closure and reduced water flow lead to physiological and biochemical changes also through the production of reactive oxygen species (ROS). Elevated ROS concentrations increase lipid peroxidation, programmed cell death and decrease photosynthetic functions due to chlorophyll degradation (Okuma et al., 2011). Similar findings have been described in nutritionally stressed plants (Molina and Covarrubias, 2019). However, the results indicated that AMS and ASA alleviated the effects of water reduction on vegetative growth and chlorophyll production. Recent reports have shown synergistic relationships between AMS and nitrogen-stimulated nitrate reductase activity, which is involved in the synthesis of nitric oxide precursor of ammonium sulphate, thus alleviating water stress symptoms by enhancing defence systems and water potential (Kaya, 2021).

Ion accumulation
Of all ions analysed, statistical analysis revealed a significant AxB interaction only for Ca 2+ content. However, it was observed that when AMS was present water-restricted and irrigate plants showed similar Ca 2+ accumulation in shoots.
The results suggest that ammonium sulphate applications could increase Ca 2+ accumulation under water deficit. The uptake and transport of dissolved Ca 2+ in the soil solution occurs by transpiration following differences in water potentials with respect to the atmosphere. Ca 2+ is an essential mineral element that acts as a cellular second messenger signaling physiological and transcriptional responses to stress conditions (Kudlak et al., 2010). It also acts by enhancing stomatal regulation and activating a number of enzymes. Recent studies have shown that nitrogen is an element that promotes stress resistance by acting synergistically with Ca 2+ accumulation (Zhang et al., 2018).

Osmoprotectants and non-enzymatic antioxidants
Although generally treated plants had higher concentration of Pro and TF, only the effect of water regime was statistically significant. At the same time, a significant between the water regime and treatment was detected for TCF. However, it was observed that plants with treatments and water restriction presented similar values in terms of TCF increases with respect to irrigated plants.
Positive results on osmoprotectant accumulation have been widely described in stressed plants of the genus Stipa (Yang et al., 2021). In the same direction, other results showed that ASA or ammonium sulphate increase proline concentration, flavonoids and phenolic compounds in Poaceae, such as Festuca arundinacea (Neuberg et al. 2010) or in dicots such as Simarouba glauca (Awate et al., 2014) and chickpea (Hussain et al., 2020).

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The above results suggest that applications of ASA or ammonium sulphate to plants under water deficit could stimulate the synthesis of osmolytes and antioxidant compounds in their cells, such as proline, flavonoids and phenolic compounds. These compounds have a high capacity to degrade reactive oxygen species (ROS) produced under stress (Farooq et al., 2009;Akinci and Lösel, 2012). Proline is a protein precursor with antioxidant activity that acts in detoxification and protection through cellular homeostasis by providing a proper redox balance. Proline also induces a number of genes responsible for the activation of antioxidant enzymes, improving the water potential in plants (Neuberg et al., 2010;Nazar et al., 2015). On the other hand, phenols and flavonoids can chelate transition metal ions, directly scavenge active molecular oxygen species and quench lipid peroxidation by trapping the alkoxide radical. In addition, flavonoids and phenylpropanoids are oxidised by peroxidase and act in the H2O2 uptake system, phenolic / AsA / POD (Kaya, 2021). Other results on different species and different experimental conditions indicate that ASA or ammonium sulphate enhance the synthesis of proline, flavonoids and phenolic compounds against water stress (Nazar et al., 2015;Kaya, 2021).

Antioxidant enzyme activity
Increases in ROS such as O2, O3 or H2O2 occur in plants under stressful conditions. These radicals produce lipid peroxidation in the cell and at high concentration may cause cell death. As a defence, organisms respond by increasing the activity of antioxidant enzymes such as SOD, CAT and APX, with SOD acting in a first line of action by catalysing the dismutation of O2to H2O2 through its neutralisation by the addition of two hydrogen ions reducing the formation of OHvia the Haber-Weiss reaction. On the other hand, CAT and APX catalyse the conversion of H2O2 to H2O and O2 alleviating the damage (Apel and Hirt, 2004;Das and Roychoudhury, 2014;Noctor et al., 2018;Farooq et al., 2020).
Of the four enzymes analysed, only higher APX activity was detected under water restriction. On the other hand, there was an AxB interaction for CAT and SOD activity. The pattern of variation in the antioxidant enzymes varies according to the species studied or the type of stress applied. In a species of the same genus, S. lagascae, increases in APX and GR but decreases in SOD activity were reported under saline conditions (Abdellaoui et al., 2017), whereas the activity of the antioxidant enzymes varied in Juncus not only in dependence of the species, but also within species according to the applied water or salt stress treatments (Al Hassan et al., 2017).
Regarding the effect of acetylsalicylic acid and AMS on antioxidant enzymes activity, there are no reports in the genus Stipa and only a few published data in monocotyledonous plants. Wheat showed higher activity of SOD, CAT and APX when treated with ASA (Hassanein et al., 2015). However, no clear trends in enzymatic activity under abiotic stresses treated with ASA have been reported. In this respect, whereas a decrease in SOD, CAT was found in Oryza sativa under water stress (Guo et al., 2007) in Spartina alterniflora were reported increases in the activity of CAT, SOD and APX with ammonium treatments (Hessini et al., 2013) and no clear trends in antioxidant enzyme activity in maize (Zhang et al., 2007).
Our findings suggest that applications of ASA or AMS could enhance SOD and CAT activity in some species under stressful conditions. SOD and CAT are enzymes in charge of systematically degrading ROS and reducing their toxic level to lower and lower levels.

Conclusions
Our findings indicate that water stress had a stronger effect on the growth of plants grown in the absence of AMS and acetylsalicylic acid or their combination. Plants treated with these compounds, or their combination had increased concentrations of proline and total flavonoids, but treated plants under water restriction showed similar values in terms of total phenolics. The activity of APX was enhanced under water restriction conditions, and an AxB interaction for CAT and SOD activity was detected. However, plants treated with acetylsalicylic acid and ammonium sulphate under water restriction showed similar reductions in the activity of both enzymes compared to irrigated/ASA.
The results obtained show that these additives can improve responses to water deficit in Stipa caudata, a species with great potential in environmental restoration or biofuel production programmes. However, several questions remain open about the relative role of ASA and AMS in other physiological and biochemical mechanisms such as the regulation of xylem water potential, photosynthetic activity and aerial dry matter production.
All authors read and approved the final manuscript.
Ethical approval (for researches involving animals or humans) Not applicable.