Nanobiotechnological Approaches to Enhance Drought Tolerance in Catharanthus roseus Plants Using Salicylic Acid in Bulk and Nanoform

Drought has a detrimental effect on crop production, affecting economically important plants’ growth rates and development. Catharanthus roseus is an important medicinal plant that produces many pharmacologically active compounds, some of which have significant antitumor activity. The effect of bulk salicylic acid (SA) and salicylic acid nanoparticles (SA-NPs) were evaluated on water-stressed Catharanthus roseus plants. The results showed that SA and SA-NPs alleviated the negative effects of drought in the treated plants by increasing their shoot and root weights, relative water content, leaf area index, chlorophyll content, and total alkaloids percentage. From the results, a low concentration (0.05 mM) of SA-NPs exerted positive effects on the treated plants, while the best results of the bulk SA were recorded after using the highest concentration (0.1 mM). Both treatments increased the expression level of WRKY1, WRKY2, WRKY40, LEA, and MYC2 genes, while the mRNA level of MPKK1 and MPK6 did not show a significant change. This study discussed the importance of SA-NPs in the induction of drought stress tolerance even when used in low concentrations, in contrast to bulk SA, which exerts significant results only at higher concentrations.


Introduction
Catharanthus roseus is an evergreen medicinal plant, belonging to the family Apocynaceae, containing many medicinal compounds. It has a long history of being used to treat a variety of diseases, including diabetes [1] and gonorrhea [2]. Vincristine and vinblastine are two alkaloids derived from the stem and leaves of this plant; they have been used to treat different types of cancer in combination with another chemotherapeutic. C. roseus contains about one hundred and twenty alkaloids, seventy of which have pharmacological importance [3]. The production of these secondary metabolites is widely affected by different biotic and abiotic stress conditions [4].
Abiotic stresses, including salinity, drought, heavy metals, and flooding, are major factors hindering crop productivity around the world. The previous investigations reported that 90% of the agricultural lands are vulnerable to these abovementioned stress factors [5]. Drought and heat are the most dangerous abiotic stress factors, impairing crop production in large areas around the world. The negative effect of droughts on plant growth is attributed to the decrease in water-soluble nutrients, which inhibits the photosynthetic process by damaging the photosynthetic pigments, thus decreasing chlorophyll and thereby The goal of this research is to use various SA concentrations in bulk and nano form to reduce the effects of drought stress on Catharanthus roseus plants. It also evaluates the effects of these concentrations on morphological, physiological, and biochemical traits, as well as stress-responsive gene expression in stressed plants.

Preparation of Salicylic Acid Nanoparticles
Salicylic acid (SA) was purchased from Sigma-Aldrich (CAS number: S5922, BioXtra, ≥99.0%). A total of 20 mg of SA was weighed and dissolved in 20 mL of absolute ethanol and sonicated with an ultrasonic power (XUBA3 Analogue Ultrasonic Bath, Grant Company, Devizes, UK) and a frequency of 50 kHz for an hour at room temperature (25 • C) [40].

Characterization of Nanomaterials Using Dynamic Light Scattering (DLS)
The distribution and particle size of SA-NPs were determined using a Zetasizer Nano ZS and a dynamic light scattering approach (Malvern Instruments, Malvern, UK). A total of 30 µL of the generated nanoparticles were diluted in 3 mL of deionized water at room temperature before testing. The particle size was estimated by taking the mean of the Z-averages of three distinct batches of each of the aforementioned nanoparticle types.

Transmission Electron Microscopy (TEM)
Transmission Electron microscopy was utilized to study the morphology of all three kinds of nanoparticles. In this case, a drop of the nanoparticles solution was sonicated before being placed on carbon-coated copper grids (CCG) and allowed to dry completely by allowing water to evaporate at ambient temperature. Electron micrographs were taken at Al-Azhar University's Regional Center for Mycology and Biotechnology (RCMB) using a JEOL GEM-1010 transmission electron microscope set at 70 kV [41].

Plant materials, Drought, and SA Treatments
The seeds were sterilized by soaking them for 3 min in 70% percent Ethanol and then rinsing them several times with sterile H 2 O. Catharanthus roseus (Cape periwinkle) seeds were sterilized and sown in plastic pots filled with a 5:1 (v/v) mixture of clay soil and peat. Treatments were administered between 45 and 60 days. Pots were exposed to drought stress alone while maintaining 25% field capacity, and other pots were exposed to drought stress as well as different concentrations of salicylic acid nanoparticles (0.05 mM and 0.1 mM) and bulk salicylic acid (0.05 mM and 0.1 mM) by foliar application, while other pots were irrigated to keep the soil at the appropriate moisture level or left untreated as a control, or treated with the mentioned concentrations of bulk salicylic acid and salicylic acid nanoparticles. Five pots were used for each treatment, and plants were treated with SA and SA-NPs three times with interval of five days.

Measuring Growth Parameters
The growth parameters of treated and untreated 60-day-old Catharanthus roseus plants were measured using five plants of each treatment, and the fresh weights of shoots and roots were recorded first. Then, plants were left to dry at 60 • C for 72 h to measure the dry weight of the shoots and roots and the root-shoot ratio. In addition, the leaf area index (LAI) was determined using the following formula as suggested by Watson (1947) [42]. LAI = Leaf area plant/Area occupied the plant (1)

Relative Water Content (RWC) Measurement
Leaves of treated and untreated 60-day-old plants were cut and sealed in plastic bags and their fresh weights were recorded immediately after cutting.. Then, they were soaked in distilled water for twenty-four hours at room temperature to record the turgid weight. Finally, turgid leaves were dried in an oven for 72 h at 70 • C to record their dry weight. RWC was calculated using the formula of Schonfeld et al. (1988) [43]: where FW = Fresh weight, DW = Dry weight, and TW = Turgid weight

Chlorophyll a, b and Carotenoid Content Measurement
Total chlorophyll a and b and carotenoid contents in fresh leaves from 60-day-old plants were measured using the method of Arnon (1949). One gram of fresh tissue of leaves was ground in a mortar and pestle using 10 mL of 80% acetone. The optical densities (OD) of the solutions obtained from different treatments were recorded at 663, 645, 480, and 510 nm using a spectrophotometer. The content of the three photosynthetic pigments was measured using the following formulas and values, which were expressed in mg g-1 FW [44]: Chlorophyll Chlorophyll where V = volume of the extract (mL) and W = weight of the fresh leaf (g).

Total Alkaloid Measurement
Total Alkaloid content was measured using Harborne's (1973) method. Briefly, five grams of air-dried leaves was weighed and then ground to powder. Then, it was incubated for four hours in 250 mL of 10% acetic acid in ethanol. A water bath was used to evaporate and concentrate the solution until it was one-fourth the original volume of the extract after the solution had been filtered to remove debris. Then, ammonium hydroxide was added dropwise to the concentrated extract until the precipitation process was complete and the obtained extract was allowed to settle. This step was followed by the collection and washing of precipitate using diluted ammonium hydroxide. Finally, the precipitate was filtered, dried, and weighed, and the percentage of total alkaloids was determined by dividing the obtained weight by the weight of the used air-dried leaves [45].

RNA Extraction and Gene Expression Profiling Using Quantitative Real-Time PCR (qRT-PCR)
The total RNA was extracted and purified using a total RNA extraction kit (Bioflux, Beijing, China) from the treated and untreated Catharanthus roseus leaves. qRT-PCR was performed with ABT One-Step SYBR kit (Rox) with an RT-PCR Kit, using 100 ng total RNA/reaction, RNasin Ribonuclease Inhibitor (Promega Corporation, Madison, WI, USA), and gene-specific primers (Table 1). Triplicate PCR reactions, as well as RNA template negative and non-template controls, were carried out and used. Each reaction contained 2.5 µL of RNA (except for NTC), 10 µL of 2× reaction buffer, 0.8 µL of superscript III-RT platinum Taq mix, 0.5 µL of SYBR green dye, and 0.3 mM of each forward and reverse primer of the seven studied genes ( Table 1). The final volume was adjusted to 20 µL by adding RNase-free water. Reactions were measured using Prime Q real-time qPCR equipment (Techne, London, UK) with a one-step cycling protocol at 45 • C for 10 min, 95 • C for 10 min, 45 cycles of 95 • C for 15 s, and then 60 • C for 30 s followed by 72 • C for 30 s. For qPCR data normalization, the actin gene (GenBank MG813871.1) was used as an internal reference gene. All experimentally generated variations in the expression of the genes under investigation are represented as n-fold changes in comparison to the controls. The formula RQ = 2 -∆∆CT was used to calculate the relative gene expression ratios (RQ) between the treatment and control groups [46].

Statistical Analysis
Analysis of variance (ANOVA) was carried out using SPSS version 19 (SPSS Inc., Chicago, IL, USA). LSD test was conducted to test the significance between mean values (p < 0.05).

Characterization of SA Nanoparticles
The prepared SA nanoparticles were characterized by different methods to analyze their particle sizes and aggregation states. A dynamic light scattering technique was performed to understand the stability and the size distribution of the prepared SA-NPs. A TEM (transmission electron microscope) analysis showed that the SA-NPs were widely dispersed and composed of particles with an average of <100 nm. Moreover, the TEM micrograph showed that the SA-NPs had a spherical morphology with an average size between 5.71 and 17.3 nm (Figure 1a). The zeta potential results for the SANPs showed a positive surface charge with a value of approximately 3.99 mV (Figure 1b). The analysis showed that the size distribution range was within 20 nm and the curve showed a monomodal distribution (polydispersity index PDI), as shown in (Figure 1c).

Plant growth Response
Drought stress (25% field capacity) significantly (p < 0.05) affected Catharanthus roseus's growth parameters in terms of plant height, shoot and root dry weight, and leaf area index. The results showed that all these parameters had improved by spraying the plants with all the studied concentrations of SA and SANPs under drought and normal irrigation conditions (Table 2). Under drought stress, both treatments increased the plant

Plant Growth Response
Drought stress (25% field capacity) significantly (p < 0.05) affected Catharanthus roseus's growth parameters in terms of plant height, shoot and root dry weight, and leaf area index. The results showed that all these parameters had improved by spraying the plants with all the studied concentrations of SA and SANPs under drought and normal irrigation conditions ( Table 2). Under drought stress, both treatments increased the plant height and plant weight compared to the control. Plants treated with 0.1 mM of the SANPs recorded the highest plant height (55 cm), a 14.58% increase, compared to the untreated plants under drought stress. Whereas the highest shoot and root dry weights (128% increase) were evidenced in the plants treated with 0.05 mM of the SANPs under drought stress (2.4 and 3.35 gm, respectively) compared to the untreated stressed plants. The root-shoot ratio also decreased significantly (p < 0.05) in response to drought stress, and this harmful effect was adverted by all the studied treatments.

Root Dry Weight (gm) Root-Shoot Ratio
Normal irrigation 53.5 e,f,g ± 0.88 1.4 e ± 0.61 0.84 d,e ± 0.31 0.60 e,f ± 0.017 Each value represents the mean ± SE of species in different treatments. Small letters represent significant differences among treatments tested using the LSD test. All tests were considered significant at p < 0.05.

Chlorophyll a and b and Carotenoid Content
Drought stress significantly (p < 0.05) decreased chlorophyll a (Chl-a) content (0.75 mg/gm) compared to the control. However, using SA in both types of formulations increased the chl-a and chl-b content; the highest chlorophyll content was recorded in the plants treated with 0.05 mM of SANP at chl-a (0.96 mg/gm), a 25.33% increase, compared to the untreated plants in underwater deficiency conditions. Whereas the highest increase in carotenoid content (0.31 mg/gm) was recorded in drought-stressed plants treated with 0.05 mM of SA, which increased carotenoids 16% compared to the control (Table 3).

Relative Water Content and Leaf Area Index
Water deficiency significantly (p < 0.05) reduced the relative water content (RWC) in the plant leaves (Table 4). Under drought conditions, spraying plants with 0.05 and 0.1 mM of SA improved the RWC by 71.66% and 85.33%, respectively, while using SANPs (0.05 and 0.1 mM) improved the RWC value by 86.1% and 89.17%, respectively. The leaf area index was also negatively affected by drought stress: water deficiency decreased the leaf area in the untreated plants by 20.5% compared to the normally irrigated plants. On the other hand, the two used treatments exerted a significant effect on alleviating this inhibition. The highest increase in the leaf area index was evidenced in plants under drought stress treated with 0.05 mM of SANP (143% increase), followed by 0.1 mM of the SANP treatment (4.87), a 135.27% increase compared to the control. Each value represents the mean ± SE of species in different treatments. Small letters represent significant differences among treatments tested using the LSD test. All tests were considered significant at p < 0.05.

Alkaloid Content
The results in Table 5 show that the drought stress increased the alkaloid content (0.77) compared with its content in the normally irrigated control (0.72). The highest increase in alkaloid content was recorded in the plants treated with 0.05 mM SANP, which increased alkaloid content by 0.83 followed by 0.82 in the plants treated with 0.1 mM SA compared to the untreated plants under drought stress.

Effect of Different Treatments on Expression Profiling of Drought Tolerance Associated Genes Using qRT-PCR
The expression levels of seven drought tolerance-associated genes-LEA, WRKY1, WRKY2, WRKY40, MYC2, MPK1, and MPK6-were upregulated under elicitation by the SA and SANPs at 0.05 and 0.1 mM. The results showed that the mRNA level of the LEA gene increased in the plants treated with both SA and SANPs; the highest expression increase was quantified in the plants treated with 0.05 mM SA-a 5.6-fold increase (Figure 2). In Figure 3A,B, WRKY1 and WRKY2 expression levels increased in response to SA and SANPs at both concentrations, except for the expression of the WRKY40 gene ( Figure 3C), which was highly upregulated under the effect of foliar application with 0.05 and 0.1 mM SANPs,

Discussion
Drought significantly reduced the fresh and dry weights of shoots and roots, as well as their length, according to the findings. Drought, similar to other abiotic stressors, causes cellular oxidative stress and reactive oxygen species production [47]. In low and moderate concentrations, these free radicals act as second messengers, activating plant tolerance. However, at high concentrations, reactive oxygen species are destructive to DNA, proteins, and biological membranes, and they also inhibit enzyme activity, including photosynthesis enzymes. On the other hand, the water-stressed plants treated with bulk SA acid and SA-NPs mitigated the negative effects of drought on growth parameters. These results are in line with the previous studies performed on different plants including Zea mays [48], Conocarpus erectus L. and Populus sp [49], and Rosmarinus officinalis L. [50].
In contrast to bulk SA, SA-NPs have a mitigating effect at low concentrations; however, their results were only significant at higher concentrations (0.1 mM). Salicylic acid promotes plant growth by increasing stomatal conductance and improving water relations, which promotes photosynthesis and increases plant growth [51]. Furthermore, by inducing root formation, nutrient uptake, activating photosynthetic enzymes, and cell division, SA helps plants mitigate the negative effects of moisture deficiency on growth.

Discussion
Drought significantly reduced the fresh and dry weights of shoots and roots, as well as their length, according to the findings. Drought, similar to other abiotic stressors, causes cellular oxidative stress and reactive oxygen species production [47]. In low and moderate concentrations, these free radicals act as second messengers, activating plant tolerance. However, at high concentrations, reactive oxygen species are destructive to DNA, proteins, and biological membranes, and they also inhibit enzyme activity, including photosynthesis enzymes. On the other hand, the water-stressed plants treated with bulk SA acid and SA-NPs mitigated the negative effects of drought on growth parameters. These results are in line with the previous studies performed on different plants including Zea mays [48], Conocarpus erectus L. and Populus sp. [49], and Rosmarinus officinalis L. [50].
In contrast to bulk SA, SA-NPs have a mitigating effect at low concentrations; however, their results were only significant at higher concentrations (0.1 mM). Salicylic acid promotes plant growth by increasing stomatal conductance and improving water relations, which promotes photosynthesis and increases plant growth [51]. Furthermore, by inducing root formation, nutrient uptake, activating photosynthetic enzymes, and cell division, SA helps plants mitigate the negative effects of moisture deficiency on growth.
Water deficiency decreased chlorophyll content in this study; the decrease in the photosynthetic pigments in the stressed plants had previously been demonstrated by Munne-Bosch and Alegre (2000) [52] in different species. This effect was caused by the action of released reactive oxygen species on the thylakoid membrane; chlorophyll degradation by chlorophyllase, which is activated in drought-exposed plants; and the decreased activity of chlorophyll synthase [53,54]. In water-stressed plants, salicylic acid reversed this effect, and the chlorophyll content increased significantly. These findings are attributed to salicylic acid's protective role in preserving plant membranes, including the thylakoid membrane, by activating antioxidant enzymes and non-enzymatic antioxidants.
Furthermore, salicylic acid activates photosynthetic enzymes while inhibiting chlorophyll oxidase, allowing chlorophyll degradation to continue. Our findings are consistent with those of Ghazali and Estaji (2020), who conducted research on Capsicum annuum [55].
Many plants showed a decrease in the leaf area index in response to water deficiency [56][57][58][59]. The leaf area is affected by assimilates and turgor pressure, both of which reduce water stress. In the presence of moisture stress, various plants tend to reduce the area of young leaves and the expansion of developed leaves to reduce transpiration. Salicylic acid, on the other hand, increased leaf area index in both water-stressed and well-watered plants. Salicylic acid increased the leaf area index by increasing the turgor pressure via assimilates, proline, and soluble proteins' accumulation [50].
The results revealed a significant decrease in the relative water content in plants subjected to drought stress without the application of salicylic acid. These findings are consistent with the findings of Khazei and Estaji (2020) [55], who discovered that drought significantly reduced the relative water content. This reduction could be due to an increased membrane permeability and a decreased water uptake. RWC is a good indicator of drought resistance; in other words, plants with a high RWC should have higher yields under drought stress [60].
In all treatments, the total percentage of alkaloids increased when compared to the control, with the highest recorded value obtained when treating plants with the highest concentration of salicylic acid nanoparticles with drought stress (0.82 percent) when compared to untreated plants under drought stress (0.77 percent). Similar findings were reported by Ababaf et al. (2021) [61], who investigated the effect of salicylic acid and a combination of salicylic acid and jasmonic acid on a drought-stressed C. roseus plant. Under drought conditions, the expression of alkaloid biosynthesis enzymes such as strictosidine synthase, tryptophan decarboxylase, and deacetylvindoline-4-Oacetyltransferase increases, resulting in an increase in alkaloids.
In addition to inducing stomatal closure to prevent further water loss in plants under drought stress, salicylic acid treatment induces the production of secondary metabolites that scavenge reactive oxygen species to protect the plants from lipid peroxidation and other oxidative damage-related harmful effects [64]. Secondary metabolite production is modulated by various transcription factors such as WRKY, bHLH, and MYB, which are induced by stress conditions, plant hormones such as salicylic acid, MAPK, and reactive oxygen species to ultimately affect the expression of secondary metabolites related to genes [65].
Plant gene expression and associated biological pathways can be altered by nanoparticles, affecting plant growth and development [66]. The results showed that the foliar application of SA at 0.05-and 0.1-mM increased LEA gene expression in plants under drought stress. The induction of the dehydrin gene, one of the LEA genes, in response to salicylic acid was previously observed in drought-stressed barley plants treated with 0.2 mM salicylic acid [67]. LEA is a plant protein class that accumulates in response to environmental stress and serves specific protective functions in plant cells. Dehydrins have been shown in vitro to be capable of membrane stabilization, protein aggregation prevention, cryoprotection, nucleic acid protection, ROS scavenging, and the binding of small ligands such as water, ice crystals, and metal ions [68].
Multiple WRKY genes contribute to the drought stress response via distinct signaling pathways, making these genes one of higher plants' largest transcription factor families [28,29]. The current study found that SA and SA-NPs induced the expression of WRKY1 and WRKY2 genes in plants under drought stress at both concentrations. While a field application of 0.05 and 0.1 mM SANPs on drought-stressed plants increased WRKY40 mRNA levels compared to negative control plants, untreated drought-stressed plants did not experience the same increase. According to Gao et al. (2018), WRKY2 plays an important role in the stress response, and the TaWRKY2 promoter contains cis-elements involved in plants' signaling of stress-related hormones such as methyl jasmonate acid (MeJA) and salicylic acid (SA) [69].
SA can both stimulate and inhibit the expression levels of WRKY70 transcription factor [30]. Furthermore, it regulates target gene expression by identifying and binding W-box cis-elements or interacting with other regulatory factors, and thus plays an important role in plant growth and development, abiotic stress tolerance, and other biological processes [32,34,70]. Pathogens or salicylic acid were found to induce WRKY gene expression in Arabidopsis [71]. The overexpression of ZmWRKY40 in Zea mays reduced the ROS content and increased the activities of POD and CAT under drought conditions, as well as activating the expression of the stress-responsive genes STZ, DREB2B, and RD29A [72].
In general, WRKYs mediate the salicylic acid response to stress-enhancing growth parameters, with plants overexpressing these transcription factors showing an increase in their roots' dry weight [73].
Furthermore, different treatments increased the mRNA level of the MYC2 transcription factor gene, with the highest fold change recorded in the plants treated with 0.1 mM of SA-NPs (5.1-fold increase). When compared to the negative control, the same concentration of bulk salicylic increased it by 2.4-fold. MYC2 is an ABA-related gene that belongs to the bHLH transcription factor family, which confers stress tolerance in plants. Salicylic acid stimulates H 2 O 2 accumulation in the early stages of drought (0-6 days), followed by H 2 O 2stimulated ABA accumulation and the induction of ABA signaling and responses [74].
In comparison to the negative control, the foliar application of SA and SA-NPs at 0.05 and 0.1 mM slightly increased the expression of MPK1 and MPK6 genes in the plants under drought stress. MAPK transcription factors play critical roles in the regulation of stressrelated hormone signaling, the activation of stress-related responses, tissue differentiation, and metabolism [75]. Furthermore, it has been demonstrated that the upregulation of MAPKs induces stress tolerance [76].
The results showed that the SA-NPs exerted significant effects even at very low concentrations when compared to the bulk material due to their greater solubility, surface area, reactivity sorption capacity, and controlled-release kinetics to targeted sites, making them a "smart delivery system." All of these properties increase input efficiency, reduce relevant losses, and reduce environmental pollution [26], especially when the nanoparticles used are produced naturally in plants, such as salicylic acid. Nanoparticles stimulate proline production and protect membranes from damage. Several studies have also shown that nanoparticles can activate stress-related genes ( Figure 6) [77].

Conclusions
The foliar spraying of in C. roseus with bulk SA and SA-NPs was found effective in reversing the negative effects of water stress. The foliar application To deal with both biotic and abiotic stress, nanoparticles may stimulate the production of secondary metabolites. They enter plant cells, causing membrane damage and the production of reactive oxygen species, which activates antioxidant production and mitogenactivated protein kinase, inducing secondary metabolite production genes ( Figure 6) [78].

Conclusions
The foliar spraying of in C. roseus with bulk SA and SA-NPs was found to be very effective in reversing the negative effects of water stress. The foliar application of SA-NPs, especially at 0.05 mM, improved growth, photosynthetic pigment biosynthesis, and cellular osmotic adjustment. Water stress tolerance was also associated with antioxidative defense mechanisms, processes such as nutrient uptake enhancement, the induction of root formation, and increased cell division in the apical region of the root meristem; ultimately, it results in an increase in plant growth and an increase in secondary metabolites such as alkaloids to deal with stress conditions. Furthermore, SA increases the expression of transcription factors and genes involved in stress tolerance. Therefore, foliar sprays containing bulk SA and SA-NPs were very effective in mitigating the negative effects of water stress in C. roseus.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author. The data are not publicly available due to privacy concerns.