Ascorbic acid and Proline alleviat the adverse effects of salinity stress in Duranta erecta L. var. Variegata plants


 The present study was carried out at the Faculty of Desert Agriculture and Environmental in Fouka, Matrouh University (Matrouh governorate) Egypt, during the two successive seasons of 2019/2020 and 2020/2021. This investigatin aimed to study the effects of ascorbic acid (0.0, 100, 200 ppm) and proline (0.0, 40, 80 ppm) on alleviating the adverse effects of salinity stress at 0.0, 2000, 4000, 6000 ppm seawater in Duranta erecta L. var. Variegata plants. The experiment was designed in a split-plot design in random completely block design (RCBD) with three replications. In this experiment, the main plot was salt water levels and the sub-plot was spraying by proline and ascorbic acid treatments. The results showed that treated plants with proline at 80 ppm and ascorbic acid at 200ppm enhanced plant height, leaves dry weight, leaf green color degree (SPAD units), plant survival percentage, stomata measurement, potassium of leaves and antioxidant such as superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), cytochrome b5, cytochrome P450 (CYP450), amidopyrine N-demethylase and aniline 4-hydroxylase activity. Moreover, treatments deacreased thiobarbituric acid reactive substances (TBARS), sodium and chlorid. Finaly, proline and ascorbic acid alleviate the negative effects of salinity and increase Duranta plant growth and plant survival percentage by inhancing the antioxidant enzymes, potassium cations and photosynthetic system, decresing DNA breakage %, harmful ions eg:sodium and chloride.


Introduction
Duranta erecta L. var. Variegata, belongs to the family Verbenaceae, is a small tree or a vigorous shrub and a sprawling shrub. It is planted frequently for the beauty of white and green leaves and blue tubular owers appearances almost all year round (Huxley et al. 1992). It is grow in tropical and subtropical areas and used for accent plants (Pipattanawong et al. 2008).
Salinity reduces plant growth, exposure of plants for long periods to salinity or severe salinity leads to death of plants. Salinity is a major concern in arid and semi-arid regions, especially the region that have not enough amount of rain to wash the soil from salts, and propten exacerbated by climate change (Ramawat 2010). Salinity also modi es gene expression (Soussi et al. 2001) increased the levels of reactive oxygen species (ROS) (Apel and Hirt 2004), chlorophyll breakdown (Kato and Shimizu 1987) and many other effects such as leaf senescence (Allu et al. 2014). Gene expression leads to the emergence of new proteins that are not present before the stimulation process. It is known that in recent time's fresh water resources have been greatly depleted, so it is necessary to try to use different alternatives sources, such as desalinated water or ground water that suffers from salinity. Salinity problems have become more prevalent in the past ten years, using of alternative non-potable water become a major contributor to irrigation (Marcum 2006). From the above, it is important to choose tolerant plant varieties and species or search about some subatances can help plants to tolerat ahigh levels of salinity (Hassanain et al. 2017). Several enzymes help detoxify reactive oxygen species. Superoxide dismutase is the rst defense enzyme that converts superoxide into H2O2 (Sairam et al. 2005).
Ascorbic acid (C 6 H 8 O 6 ) is a small molecular soluble in water and insoluble in other solvents as chloroform, benzene and fats. Ascorbic acid acts as an antioxidant, working to remove enzymatic toxins such as H 2 O 2 and other harmful factors that affect plant photosynthesis (Galal et al. 2000). It is a reductant and reacting with and/or scavenging many types of free radicals. Ascorbic acid reacts non-enzymatically with superoxide, hydrogen peroxide and singlet oxygen (Pourcel et al. 2007). Proline is an amino acid that preserves the plant cell under conditions of drought and salinity where it prevents and reduces the breakdown of protein in the cell and maintains the vitality of the plant. Proline acts as a visceral osmotic solute and as a source of energy and nitrogen, so proline has a role in reducing the harmful effects of salt stress (Venkatesan and Chellappan 1998).
This investigation aimed to study, the effects of ascorbic acid and proline on alleviating the adverse effects of salinity stress in Duranta erecta L. var. Variegata plants.

Material And Methods
The present study was carried out during two successive seasons of (2019-2020) and (2020)(2021) at the Faculty of Desert Agriculture and Environmental in Fouka, Matrouh University (Matrouh governorate) Egypt, to investigate the effects of proline and ascorbic acid on the growth, some chemical and biochemical composition of Duranta erecta L Var. Variegata plants under salinity stress condition. One year old plants were obtained from commercial (private) nursery for ornamental plants in Marsa Matrouh city. The plants had a uniform sizes and shapes, with 5 branches / plant and 70 cm in height were used. One plant / pot was cultivated. The used soil is a mixture of 50% sandy soil (the available soil in the farm of Faculty of Desert Agriculture and Environmental in Fouka, Matrouh University and 50% clay soil. Physical and chemical soil analysis is shown in Table (1). Treatments and cultural aspects: four concentrations of the seawater irrigation (tap water, 2000, 4000 and 6000 ppm), and two anti -salt stress materials: proline at 40 and 80 ppm and ascorbic acid at 100 and 200 ppm were used. Plants were sprayed with anti-salt stress materials and spraying with tap water as a control until run off points. Duranta plants were planted in pots of 30 cm diameter on 20th of December 2019 in the rst season and 20th of December 2020 in the second season. Plants sprayed directly with anti-stress materials repeated every two weeks for 4 months. Irrigation was carried out twice a week with saline water and irrigated with tap water once every two weeks. Plants were irrigated three times a week from May to the end of the experiment (20 August in both seasons) with saline water and irrigation with tap water once every two weeks until 20th of August. On the other hand, spraying plants with anti-salt stress compounds was stoped after the rst cut (four months fom the beginning of the experiment) while irrigation with seawater concentration was continued to the end of the experiment. Hco 3 -( meq L -1 ) 4.7 Cl -( meq L -1 ) 90.1 SO4-( meq L -1 ) 38.5 Ca ++ ( meq L -1 ) 36.8 Na + ( meq L -1 ) 65.2 Mg ++ ( meq L -1 ) 28.4 K + ( meq L -1 ) 2.9 Recorded data data was collected twice per season, after four and eight months for the rst and second cut respectively.
Plant height (cm) was measured.
Leaves dry weight per branch (g) leaves dry weight was estimated after dried on oven at 70 o c for 72 hours.
Plant survival was estimated as the percentage of plants survival (how many plants kept growing to that failed).

Leaf green color degree (SPAD units)
It was determined on the fresh leaves as indicator to chlorophyll content. The determination was done according to the method described by Yadawa (1986) using Minolta SPAD Chlorophyll Meter model-502.

Stomata measurements
Number, length and diameter of stomata were calculated as the methods of Dore (1986). Individual leaves (4th leaf) were rst removed from stem. Leaf a baxial epidermis cleaned using a degreased cotton ball and then carefully smeared with nail varnish in the mid-area between the central vein and leaf edge for approximately 20 min. Thin lm was peeled off from the leaf surface, placed into a microscope slide after one drop of tap water was added then the cover glass closed. Under a light microscope, number of stomata was counted, as well as length and diameter of three stomata per leaf were measured magnifying by 100 ×10 (objective × oculars). Values obtained were multiplied by 1.25; a coe cient obtained by adjusting the ocular micrometer.
Potassium contents of leaves (%) Potassium percentage was determined photometrically in the acid digested samples by using am photometer as described by Page et al. (1982).

Sodium contents of leaves (%)
Sodium percentage was determined according to Piper. (1967).
Determination of antioxidant enzymes:

Enzyme extraction
Leaves were taken to estimate the enzymes. Once after 120 days and once again after 240 days from treatment. Sample preparation was done as described by Mukherjee and Choudhuri (1983). A fresh sample (250 mg) was frozen in liquid nitrogen and nely ground by pestle in a chilled motor, the frozen powder was added to 10 ml of 100mM phosphate buffer (KH 2 PO 4 /K 2 HPO 4, pH 7.0) containing 0.1mM Na 2 EDTA and 0.1 g of polyvinylpyrrolidone (PVP), the homogenate was ltered through cheese cloth then centrifuged at 15000 g for 10 min. The supernatant was recentrifuged at 18000 g for 10 min; the resulted supernatant was collected and stored at 4 ˚C for assay of superoxide dismutase (SOD), catalase (CAT), thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), cytochrome b5, cytochrome P450 (CYP450), amidopyrine N-demethylase and aniline 4-hydroxylase activity.
Reduced glutathione(GSH) was determined according to the method described by Ellman (1959).

Determination of oxdative stress and biomarkers
Assay of cytochrome b5 and cytochrome P450 were estimated by the method of Omura and Sato (1964).
The activity of amidopyrine N-demethylase was measured according to Nash (1953).
The activity of aniline 4-hydroxylase was measured according to Kato and Gillette (1965).
The diphenylamine DNA fragmentation percentage assay is a very useful method for measuring apoptosis by determining the percentage of fragmentation of DNA into oligosomal-sized fragments. Plants DNA breakage was conducted using the procedure of Wu et al. (2006).

Experimental layout and statistical analysis
The experiment was designed in a split-plot design in RCBD with three replications. In this experiment, salt water levels expressed as the main plot and the sub-plot was spraying by proline and ascorbic acid treatments. Each experiment was included 20 treatments, 4 levels of salt-water irrigation and 5 treatments of anti-stress compounds (control, 2 rates proline and 2 rates ascorbic acid). 4 pots for each treatment per replicate and each pot contained 1 plant). The collected data in this study were subjected to analysis of variance (ANOVA) using CoStat program. Least signi cant difference (LSD) was used at 0.05 level of probability to test differences between treatments (Gomez and Gomez. 1984).

Vegetative traits
All salinity concentrations decreased signi cantly plant height and leaves dry weight per branch in both seasons and cuttings without using anti-stress compounds as shown in Table (2). Plant irrigation with 6000 ppm treatment gave the lowest values and died befor second cut. While, addition of proline and ascorbic acid at all concentrations inhanced plant growth and increased signi cantly plant height and leves dry weight in both seasons and cuttings. The concentrations of 80 ppm proline gave the best results it increased plant height by 8.0, 18.0, 9.4 and 18.44% for the rst and second cut of the rst and second seasons respectively. In addition, it increased leaves dry weight per branch by 94.4, 131.8, 96.3 and 131.3% for rst and second cut of the rst and second seasons respectively.

Chemical composition
Seawater irrigation at all levels of salinity dereased signi cantly total chlorophyll content (SPAD unit) and potassium of leaves % in the absence of proine and ascorbic acid in the both cutting and seasons as shown in Table (3). Seawater at 6000 ppm gave the lowest vaues in the rst cut of both seasons. On the other hand, all concentrations of ascorbic acid and proline increased signi cantly total chlorophyll content and potassium of leaves % in both cuttings of both seasons. Spraying plants with proline at 80 ppm gave the highest values followed by ascorbic acid at 200 ppm compared to untreated plants as the mean effect for total chlorophyll content. This increment valued by 23.4, 29.5, 26.92 and 12.9% in the rst and second cut of the rst and second season. For potassium of leaves % the increment valued by 98.3, 56.68, 85.7 and 107.1% for the rst and second cut of the rst and second seasons respectively compared with untreated plants.
Spraying plants with all concentrations of ascorbic acid and proline decreased signi cantly sodium and chloride of leaves in both seasons and cuttings. Proline at 80 ppm gave the best results. When plants irrigated with seawater at 6000 ppm and sprayed with 80 ppm proline, sodium of leaves decreased by 25.0 and 12.7% compared with unsprayed plants in the rst and second seasons of the rst cut. In addition, chloride of leaves decreased by 10.5, 15%.
Number, length and width of stomata: Table (5) showed that number, length and width of stomata were decreased signi cantly with increasing salinity level in both cuttings in most cases in the absence of anti-stress compounds. The level of 6000 ppm seawater gave the lowset values in the rst cut and plants died before second cut when the foliar spray with ascorbic acid or proline was stoped. Spraying plants with proline and ascorbic acids increased signi cantly all stomata parameter. The concentration of 80ppm proline gave the best results 12.88, 29.12 and 24.11 for number, length and width of stomata in the rst cut compared to control(9.25, 25.47 and 20.83 respectively) followed by 200 ppm ascorbic acid.
. Values in the same column not followed by the same letter are signi cantly different at the 5% level of probability. Values in the same column not followed by the same letter are signi cantly different at the 5% level of probability. LSD at 5% S = 3.29 ns F = 2.24 *** (S) * (F) = 3.88 ns S = 3.99 ns F = 1.54 *** (S) * (F) = 2.66 * Values in the same column not followed by the same letter are signi cantly different at the 5% level of probability. Values in the same column not followed by the same letter are signi cantly different at the 5% level of probability. LSD at 5% S = 1.12 ns F = 0.41 *** (S) * (F) = 0.70 ns S = 0.33 ns F = 0.35 *** (S) * (F) = 0.60 ns Values in the same column not followed by the same letter are signi cantly different at the 5% level of probability. Table 10 Effects of ascorbic acid and proline and their interaction treatments on amidopyrine N-demethylase µmol/min × kg (plant tissue) and aniline 4hydroxylase activity µmol/min /mg protein of Duranta erecta L Var. Variegata plants under salinity stress condition at 2019/2020 and 2020/2021 2019/2020 season 2020/2021 season Saline water level (ppm) Proline (P) and Ascorbic (As) concentration (ppm) Proline (P) and Ascorbic (As) concentration (ppm) LSD at 5% S = 0.04 ns F = 0.04 *** (S) * (F) = 0.08 ns S = 0.08 ns F = 0.03 *** (S) * (F) = 0.05 ns Values in the same column not followed by the same letter are signi cantly different at the 5% level of probability.
DNA breakage and Plant survival % As shown in Table (  LSD at 5% S = 13.21 *** F = 5.53 *** (S) * (F) = 11.07 ** S = 9.56 *** F = 5.53 *** (S) * (F) = 11.07 ***  (2019) on Petunia×atkinsiana any further reduction in stomatal conductance and photosynthetic rate inhibit plant growth and reduce productivity (Tester and Davenport 2003 unbalanced as a result of other components interfering with excessive concentrations of Na and Cl ions in the soil. The increase in Na + absorption caused by salinity was accompanied by a decrease in K + concentration, indicating an apparent antagonism relationship between K + and Na+ (Cuinet al. 2009).
Spraying with Proline reduces sodium absorption and enhances potassium absorption (Abd El-Samad 2010) on maize. On the other hand, Ahmed (1996) concluded that, application of ascorbic acid enhancement the amount of organic acids excreted into the soil by the roots, hence increasing the solubility of most nutrients that dissolve slowly into the rhizosphere zone. These study agreement with Wuet al. (2017)  Ascorbic acid serves as a main substrate in the cyclic pathway for enzyme detoxi cation of hydrogen peroxide (Shalata and Neumann 2001). SOD reduced ROS production, thus reducing cell oxidation, these results are agreement with Solimanet al. (2015).
In this study, treated plants with ascorbic acid and proline increased cytochrome b5 in leaves. The enzyme is a component of an ER-associated redox chain that delivers electrons to cytochrome b5, an intermediate electron donor in a variety of lipid modi cation processes including as NADH-dependent fatty acid desaturation, sterol precursor desaturation, and fatty acid hydroxylation (Rahier et al. 1997). Spraying plants with proline and ascorbic acid increased CYP450.
CYP450 is a plant enzyme that is involved in the manufacture of secondary metabolites such as phenylpropanoids, terpenoids, alkaloids, and lignins, as well as signalling molecules, fatty acids, lipids, defense-related chemicals, and plant hormones as reported by Goujon et al. (2003). However, pretreatment with proline and ascorbic acid reduced the effect of free radicals and helped maintain levels of the enzymes such as amidopyrine N-demethylase in plants.
Amidopyrine N-demethylase is an enzyme of the cytochrome P450 that helps in the metabolism of cells as reported by Marabini et al. (1994).
In this study treated Duranta erecta L Var. Variegata plants with proline and ascorbic acid increased aniline 4-hydroxylase. Because the activity of aniline 4hydroxylase is induced by an H 2 O 2 signal generated by various environmental stresses. Aniline 4-hydroxylase are important for the AOS-scavenging system, the investigation on aniline 4-hydroxylase has seldom been reported (Sawada et al. 2006).
Finally, proline and ascorbic acid increased antioxidant enzyme systems to protect plant cells from oxidative enzyme and decreased the toxic ions, DNA breakage % and increased photosynthetis, growth and survivability (%).

Conclusions
Using proline at a rate of 80 ppm or ascorbic acid at a rate of 200 ppm for four months was recommended to get the best growth of Duranta erecta L. var. varigata plants and increased antioxidant enzyme system, which irrigated with seawater concentrations. Plants irrigated with 6000 ppm seawater need to continuesly spraying with proline at 80 ppm to keep alive.

Declarations
Funding