**Horticultural Plants Facing Stressful Conditions - Ways of Stress Mitigation**

Editors

**Agnieszka Hanaka Małgorzata Majewska Barbara Hawrylak-Nowak**

Basel • Beijing • Wuhan • Barcelona • Belgrade • Novi Sad • Cluj • Manchester

*Editors*

Agnieszka Hanaka Maria Curie-Skłodowska University in Lublin Lublin Poland

Małgorzata Majewska Maria Curie-Skłodowska University in Lublin Lublin Poland

Barbara Hawrylak-Nowak University of Life Sciences in Lublin Lublin Poland

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Horticulturae* (ISSN 2311-7524) (available at: https://www.mdpi.com/journal/horticulturae/ special issues/Horticultural Plants Stressful Conditions).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

Lastname, A.A.; Lastname, B.B. Article Title. *Journal Name* **Year**, *Volume Number*, Page Range.

**ISBN 978-3-0365-9444-6 (Hbk) ISBN 978-3-0365-9445-3 (PDF) doi.org/10.3390/books978-3-0365-9445-3**

Cover image courtesy of Małgorzata Majewska

© 2023 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons Attribution-NonCommercial-NoDerivs (CC BY-NC-ND) license.

## **Contents**

Reprinted from: *Horticulturae* **2022**, *8*, 392, doi:10.3390/horticulturae8050392 ............ **141**

## **About the Editors**

#### **Agnieszka Hanaka**

Agnieszka Hanaka works as an Associate Professor in the Department of Plant Physiology and Biophysics, Faculty of Biology and Biotechnology at the Maria Curie-Sklodowska University (UMCS) in Lublin, Poland. She earned a Master of Sciences degree in Biology (specializing in biochemistry) in 1997 and a Ph.D. in Biological Sciences (specializing in plant physiology) in 2007. She obtained a post-doctoral degree (habilitation) in biological sciences in biology in 2019. The aim of the study is to examine the physiological and phytochemical responses of plants to abiotic stressors (e.g., excess metals and temperature), which are altered by the presence of signaling molecules and plant growth-promoting microorganisms. This research focuses on exploring changes in the synthesis of primary and secondary metabolites of plants treated with various environmental factors.

#### **Małgorzata Majewska**

Małgorzata Majewska works as an Assistant Professor in the Department of Industrial and Environmental Microbiology, Faculty of Biology and Biotechnology at the Maria Curie-Skłodowska University (UMCS) in Lublin, Poland. She earned a Master of Sciences degree in Biology (specializing in microbiology) in 1996 and a Ph.D. degree in Biological Sciences (specializing in microbiology) in 2003. She obtained a post-doctoral degree (habilitation) in biological sciences in biology in 2018. The aim of the study is to identify the rate and efficiency of the mobilization of cadmium previously immobilized in the root biomass and Cd immobilization via components of the soil. This research focuses on the bioremediation of the soil contaminated with heavy metals, microbial interactions in the rhizosphere and plant growth-promoting microorganisms in metal-induced stress.

#### **Barbara Hawrylak-Nowak**

Barbara Hawrylak-Nowak works as an Associate Professor in the Sub-department of Plant Physiology and Biochemistry, Department of Botany and Plant Physiology at the University of Life Sciences in Lublin, Poland. She earned a Master of Sciences degree in Biology (specializing in environmental biology) in 2000, as well as in Biotechnology in 2002 and a Ph.D. degree in Agricultural Sciences (specializing in plant physiology) in 2004. She obtained a post-doctoral degree (habilitation) in agricultural sciences in horticulture in 2017. The aim of the study is the application of trace elements and other phytoprotectants in the mitigation of the impact of unfavorable environmental factors on plants. This research focuses on the biological activity of trace elements in plants, especially grown under stressful conditions, as well as the mineral nutrition of plants, enhancement of their secondary metabolism and ecological investigations.

## **Preface**

In recent years, numerous studies have investigated the effects of various stressors on crop plants and their ability to tolerate biotic and abiotic stresses using different substances. This Special Issue provides the latest research and information on stress-induced changes in horticultural plants, as well as the biological actions, effectiveness and principles of mitigating factors in different cropping systems. A deeper understanding of the traits associated with improved plant stress resistance can accelerate the practical implementation of these advancements in enhancing crop yields.

We sincerely appreciate the authors for sharing their expertise, experience and critical discussion in preparing and revising their interesting articles in a timely manner. Moreover, we are thankful to the reviewers for their valuable suggestions and insightful remarks that significantly contributed to enhancing the quality of each manuscript. We trust that this Special Issue will be useful to those readers who are interested in stress physiology and resistance of horticultural plants.

> **Agnieszka Hanaka, Małgorzata Majewska, and Barbara Hawrylak-Nowak** *Editors*

## *Editorial* **Horticultural Plants Facing Stressful Conditions—Ways of Stress Mitigation**

**Agnieszka Hanaka 1,\*, Małgorzata Majewska 2 and Barbara Hawrylak-Nowak 3**

#### **1. Introduction**

Rapidly progressing climate change is increasing the frequency and severity of drought and salinity stresses, which are the major factors affecting crop production and the quality of ornamental plants, fruits, and vegetables. Natural acclimation and adaptation mechanisms of plants may not be sufficient to cope with these swift climate changes, and plant yield may be reduced and have a lower quality. For this reason, it is necessary to look for ways to reduce the negative effects of stress to provide constituents of the human diet in sufficient quantity and quality. Scientists are investing efforts toward the development of new cultivation techniques and improvements in the nutritional and pro-health value of fruits and vegetables.

The present treatise examines selected topics relating to stressful conditions and ways of its mitigation. The findings of these studies is especially important in understanding the molecular, metabolic, and genetic basis upon which to increase the plant resistance to environmental stresses. This Special Issue comprises twelve research papers and one review. The review article focused on the influence of salt stress on plant growth and methods of its reduction. The presented research studies were conducted at the morphological, anatomical, physiological, and molecular levels. The authors described the impact of stress caused by salinity [1–7], high temperature [8,9], drought [9], spring frosts [10], copper [11], asbestos [12], and biotic stress [13]. Different plant species were studied, i.e., sunflower, zinnia, orchid, buttercup, tomato, snap bean, lettuce, jojoba, grapevine, strawberry, and apple. This Special Issue is dedicated to the following topics: (1) the application of exogenous compounds and bioinoculants to increase stress tolerance [1–7,9,13], (2) the improvement of fruits and vegetables quality and their nutritional value [2,7,10], and (3) the planting of ornamental plants in polluted areas [1,11,12]. Interestingly, Markulj Kulundži´c et al. [8] showed that daily changes in temperature and light intensity can induce unfavorable changes in the process of photosynthesis. They observed that morning environmental conditions were optimal for photosynthetic responses of sunflower in the flowering stage, while the afternoon was characterized by elevated temperatures and excess light intensity.

#### **2. Exogenous Compounds and Bioinoculants Increasing Stress Tolerance**

Various studies indicated that application of exogenous compounds can serve as an alternative to mitigate salt stress in commercial cultivars. Eisa et al. [1], El-Beltagi et al. [5], and Annadurai et al. [9] found that melatonin application decreased the negative effect of salt, drought, and high-temperature stresses, respectively. They recognized that melatonin, as a novel biostimulator, has potential in scavenging reactive oxygen species through

**Citation:** Hanaka, A.; Majewska, M.; Hawrylak-Nowak, B. Horticultural Plants Facing Stressful Conditions—Ways of Stress Mitigation. *Horticulturae* **2023**, *9*, 1146. https://doi.org/10.3390/ horticulturae9101146

Received: 27 July 2023 Accepted: 7 August 2023 Published: 19 October 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

1

increased antioxidant activity, which shields the photosynthetic membrane from damage and therefore helps toward stress mitigation. Melatonin was found to have a positive effect at the morphological, physiological, and biochemical levels. In accordance with expectations, plant growth [1,9], fruit yield [9], photosynthetic rate [9], the activity of antioxidant enzymes, such as superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, and glutathione reductase [1,5,9], and the contents of free proline [1,5], chlorophyll, carotenoids [1,5,9], and total soluble sugars [5] were increased in stress- and melatonintreated plants. Moreover, El-Beltagi et al. [5] demonstrated that a combined treatment of melatonin and putrescine could enhance resistance to salinity in snap bean seedlings.

Aboryia et al. [4] evaluated the effect of foliar-applied proline on the enhancement of jojoba tolerance to salt stress. The physiological responses to proline treatment were manifested by an increase in the content of chlorophylls, carotenoids, minerals (N, P, K, Na, and Cl), and phenols in leaves, as well as the promotion of the antioxidant system compared with the control plants. Also, the exogenous application of proline improved most morphological (e.g., shoot number, stem diameter, number, area, and weight of leaves) and anatomical characteristic of jojoba leaves in saline conditions. In turn, Sayed et al. [2] used nano-silicon fertilizer to mitigate salinity stress. They observed that nano-silicon application on the leaves of tomato plants (*Solanum lycopersicum* L. grafted on *S. pimpinellifolium* or Edkawy) enhanced shoot and root growth, fruit yield, and fruit quality. Higher levels of mineral content, gibberellic acid, abscisic acid, and proline were detected in shoots that were grafted and subjected to nano-silicon application compared to the control treatment.

Polyhydroxy steroids phytohormones, such as brassinosteroids, can also be used to improve crop salt tolerance [6,7]. Furio et al. [7] found that strawberry treated with brassinosteroids was characterized by a higher shoot and root weight as well as a higher total weight of fruits per plant. Additionally, the quality of fruits obtained in salt stress conditions after brassinosteroids application was significantly higher than in non-treated individuals. Also, El-Banna et al. [6] indicated that grapevine seedlings sprayed with brassinosteroids tolerated salt stress better by sustaining higher photosynthetic pigment concentrations, maintaining ion homeostasis and water status, and stimulating antioxidant capacity, as well as affecting the preservation of the proper leaf anatomical attributes compared to untreated plants.

Stressful conditions can also be alleviated by using bioinoculants, which can offer a crucial alternative for organic farming [13]. Bioinoculants are a group of microorganisms which promote plant growth by making essential nutrients more bioavailable, control phytopathogens and disease development (biotic stress) as biocontrol agents, or increase tolerance to pollutants (abiotic stress). Pacheco-Trejo et al. [13] described the possibility of using *Trichoderma* spp. to protect and stimulate the growth of horticultural crops. They proved that the plant signaling events triggered by *Trichoderma* spp. are of high importance in order to understand the molecular basis involving plant protection against stresses. Also, the signaling elements of the plants from *Trichoderma* perception through late defensive responses were described. The authors explained that the activation of defense in plants stimulated by *Trichoderma* spp. will lead to an increase in crop production with consequent benefits for human health and the environment.

#### **3. Improvement in Quality and Nutritional Value of Fruits and Vegetables**

Plants exposed to various stresses deliver a lower crop production and quality of edible plants. Furio et al. [7], Ozherelieva et al. [10], and Maglione et al. [3] were looking for ways to prevent the negative effects of stress and maintain the good quality of harvested fruits and vegetables. They proposed brassinosteriods [7], iodine [3], and adaptogenic preparations [10] as protective factors against stress with the ability to improve the quality of potential yields.

Furio et al. [7] demonstrated that a pre-harvest treatment with brassinosteriods promoted the growth of strawberry in normal conditions and under exposition to water or saline stresses, as well as evaluated the percentage of fruits of commercial quality. In turn, the results of Maglione et al. [3] showed that positive stress (eustress) or essential and non-essential elements can improve the nutritive values (biofortification) of lettuce. Iodine application under moderate salinity increased the amount of the bioactive compounds acting as antioxidants, e.g., polyphenols and anthocyanins, thus exercising functional effects on human health.

Ozherelieva et al. [10] estimated the suitability of the adaptogenic preparations (phytomodulator "White Pearl Universal Antifreeze" and phytocorrector "White Pearl Drip Ca + Mg") to increase the yield and quality of apple cultivar "Sinap Orlovsky". The foliar application of these products in the early spring period significantly reduced the effects caused by scald in fruits, decreased bitter pitting, increased the average fruit weight, as well as increased the amount of sucrose and L-ascorbic acid, thus improving the taste qualities of fruits.

The conducted research including brassinosteriods [7], iodine [3], and adaptogenic preparations [10] proved their validity as additional techniques in traditional cultivation technologies, thus inducing a higher production and better quality of crops.

#### **4. Planting Ornamental Plants on Polluted Areas**

Heavily polluted areas lose their suitability both as agricultural lands and as ecological lands. The accumulation of toxins limits the growth of plants and hinders their revitalization. The constant increase in contaminated areas forces us to seek new opportunities for their cleanup and re-use.

Tugbaeva et al. [11] proposed to use *Zinnia elegans* (Jacq.) in landscaping because they observed that it performs well in copper-polluted growth medium (200 μM CuSO4). The adaptation of zinnia to the excess Cu was associated with the metabolic changes in the phenylpropanoid pathway. The intensified lignification in the roots led to the accumulation of Cu in this organ and limited translocation to the shoots, which provided plant growth. An increase in the content of H2O2 and intensity of lipid peroxidation, as indicators of stress, an elevated amount of phenolic compounds, as well as the level of expression of genes encoding 4-coumarate-CoA ligase, cinnamoyl alcohol dehydrogenase, and class III peroxidase were observed under Cu treatment.

The possibility of *Epipactis atrorubens* (Hoffm.) cultivation on serpentine dumps post asbestos mining was analyzed by Maleva et al. [12]. The aim was the naturalization of regionally rare orchid species, very beautiful bright flowers, in contaminated areas. The authors observed that the leaves of orchids colonizing these dumps accumulated lipid peroxidation products, ascorbate, free proline, soluble phenolic compounds (including flavonoids), and non-protein thiols. For this reason, they recognized that non-enzymatic antioxidants increased the adaptive potential of *E. atrorubens*. They also attributed an important role to the increased mycorrhization of orchid plants growing under adverse conditions.

In addition to studying the adaptation of plants to growth in a saline environment and the response to this stress, Eisa et al. [1] also studied the possibility of alleviating stress using melatonin. The subject of the study was buttercup (*Ranunculus asiaticus* L.), highly prized as a cut flower due to its very decorative inflorescences. It was found that the foliar application of melatonin decreased the salt-induced symptoms of retarded vegetative growth, improved physiological parameters, and soothed oxidative stress, thereby enhancing stress resistance, which can be considered as an effective practice for production under stress conditions.

**Author Contributions:** Conceptualization, A.H. and M.M.; writing—original draft preparation, A.H., M.M. and B.H.-N.; writing—review and editing, A.H., M.M. and B.H.-N. All authors have read and agreed to the published version of the manuscript.

**Acknowledgments:** We gratefully acknowledge all the authors that participated in this Special Issue.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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## *Article* **Exogenous Melatonin Application Induced Morpho-Physiological and Biochemical Regulations Conferring Salt Tolerance in** *Ranunculus asiaticus* **L.**

**Eman Abdelhakim Eisa 1,2, Péter Honfi 2, Andrea Tilly-Mándy 2 and Iman Mirmazloum 3,\***

**Abstract:** This study presents the effects of exogenous melatonin application at different concentrations (50, 100, and 200 μM) on the morphological, physiological, and antioxidant defense systems of the buttercup plant under salinity stress (4.5 and 5.5 dS·m−1 EC). Expectedly, the salinity stress negatively affected the plant growth parameters, cell membrane stability, and POX enzyme activity of *R. asiaticus* compared to non-stressed plants. However, in a dose-dependent manner, exogenous melatonin foliar application decreased the salt stress-induced symptoms of retarded vegetative growth, physiological characteristics, and oxidative stress level. The results obtained, revealed the significant effectiveness of exogenous melatonin treatment at 200 μM concentration under salt stress conditions by enhancing the plant growth traits such as chlorophyll and carotenoids content, relative water content, proline content, peroxidase enzyme activity (POD), and by the decreased electrolyte leakage rate, and Na+ content, as well as delaying the emergence of flower buds under salinity stress. The salt tolerance index percentages (STI%) for all estimated characters are also calculated for all studied parameters. This study uncovered the beneficial effect of melatonin in reducing salt stress symptoms that can be used to reduce the salinity effect in ranunculus plant production.

**Keywords:** buttercup; melatonin; proline; salt stress; POD activity; STI

#### **1. Introduction**

Salinity, particularly in arid and semi-arid regions, is the main environmental concern with detrimental effects on soil and consequent agricultural output [1]. The salt-affected land area has increased and will continue to expand due to persistently unsustainable agricultural practices and climate change [2], posing a serious threat to agricultural areas that appears to be worsening [3]. As a result, 30% of farmland will be affected in the next quarter-century, and as much as 50% will be impracticable by 2050 [4]. Therefore, it is now more crucial than ever to learn how plants adapt and cope with salt stress by understanding the potential inhibition and tolerance mechanisms. In many ways, osmotic and ionic stress caused by salt exposure alters the major biological activities of many plants, including sodium ion (Na+) toxicity, nutritional imbalance, physiological water shortage, metabolic disturbances, oxidative damage, and photo-inhibition [5,6]. Commonly, the cut flowers are not suggested as crops for saline soils or water recycling systems because they are considered intolerant to high salt concentration of irrigation water and soil [7]. Salinity, in particular, can affect the quality of the blooms, shorten plants stems, and lower the production in flora crops [7–10]. In light of this, it is crucial to test more floricultural varieties for salinity tolerance from environmental and economic points of view for sustainable development.

**Citation:** Eisa, E.A.; Honfi, P.; Tilly-Mándy, A.; Mirmazloum, I. Exogenous Melatonin Application Induced Morpho-Physiological and Biochemical Regulations Conferring Salt Tolerance in *Ranunculus asiaticus* L.. *Horticulturae* **2023**, *9*, 228. https:// doi.org/10.3390/horticulturae9020228

Academic Editors: Agnieszka Hanaka, Małgorzata Majewska and Barbara Hawrylak-Nowak

Received: 16 January 2023 Revised: 6 February 2023 Accepted: 7 February 2023 Published: 8 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

*Ranunculus asiaticus*, also known as the buttercup, a member of the *Ranunculaceae* family, is an indigenous species from Asia and the Mediterranean [11]. The buttercup has delightful and odorless flowers that have a diameter of around 5 cm and come in various colors, including yellow, orange, red, pink, and white. Ranunculus's leaves are light green and grow to a maximum height of about 40 cm [12]. Due to its gorgeous inflorescences on strong and long stems, it is considered as an annual field-grown cut flower and is highly prized as a bedding or potted plant [13]. Depending on the variety, ranunculus can be propagated by tuberous roots or seeds [14,15]. Tuberous roots are more preferred since they often result in plants with earlier flowering time and more blooms per stem than seedpropagated plants [15]. A rosette of leaves with long petioles is produced from the tuberous roots of *R. asiaticus* in the fall after the first rain rehydrates the dried tissue in its natural Mediterranean habitat [16]. Six to eight fully developed leaves signal the onset of flowering, which continues through February and May. After shoot senescence, the tuberous roots are removed, stored over the summer, and sold for the following year's plantings in gardens and flowerbeds [15]. Buttercups like partial exposure to the sun and prefer moderate low temperatures (night/day regime 5–10/12–25 ◦C; optimum day 16 ◦C) and medium to high light intensities [16]. According to Rauter et al. [17], ranunculus physiological characteristics and visual growth quality declined when the EC of irrigation water raised from 0.5 to 5.5 dS·m−1, resulting in a rise in root-zone salinity. Similar observations were reported by Valdez-Aguilar et al. [18] on the sensitivity of ranunculus to saline conditions when irrigation solution EC value was raised from 2.0 to 6.0 dS·m−1.

One of the most recent strategies that tried to reduce the impact of biotic and abiotic stressors on plants is the application of plant bio-stimulators or growth hormones to promote adaptation [19]. Melatonin (MT) is the common name for N-acetyl-5-methoxytryptamine, a derivative of the essential amino acid, tryptophan, which is also a multi-regulatory biological hormone that is vital for plant and animal lives [20]. MT is reported to be involved in the circadian rhythm [21], immunological stimulation [22], antioxidant systems [23], and the seasonal cycle of reproduction [24]. MT was first detected in plants in 1995 [25]. The involvement of MT in plant growth regulation and development, including chlorophyll production and photosynthesis, callus induction, flowering, rhizogenesis, and the ageing of the leaves, has been documented [26–29]. Additionally, melatonin is also famous for its frequent application to combat biotic and abiotic stresses in plants since its ROS scavenging ability was confirmed [30,31]. Recent studies showed that MT-treated plants were more tolerant to salinity stress, had lower levels of reactive oxygen species, electrolyte leakage, and cell damage, and had higher growth parameters than untreated plants [32,33]. The positive effect of MT application for plants exposed to stress was confirmed in different species such as in watermelon [34], cucumber [28,35], melon [36], basil [37], and maize [29]. However, no such trial (melatonin application) has been conducted on *Ranunculus asiaticus* grown under salinity stress. Therefore, the present work intends to exhibit the efficacy of MT in enhancing the salinity tress tolerance of buttercup plants in terms of morphological and physiological reactions associated with oxidative stress. In this regard, the salt tolerance index (STI%) for all the studied parameters is determined to be utilized as an indicant of salt tolerance.

#### **2. Materials and Methods**

#### *2.1. Material, Cultivation Conditions, and Trial Layout*

The pot trial was undertaken in a research greenhouse of the Floriculture and Dendrology Department at The Hungarian University of Agriculture and life science (MATE) (Budapest, Hungary). The planting process started on 1 October 2021, and the harvest occurred 150 days later. The average day and night temperature of the glasshouse was 20–15 ◦C, with 60% relative humidity. Healthy seedlings (30-days old) of *R. asiaticus* with four to five true leaves (size, 5–6 cm) were transplanted into ø (9 × 9 × 10) cm plastic pots; one plant per each pot and 135 plants per trial. The design of the experimental groups was a randomized complete block design (RCBD) in a split-plot arrangement; the main plots

consisted of three different salinity levels: (1) the control plants typically watered with 80% of the field's capacity; no MT; no salt treatment; (2) salinity treatment at EC 4.5 dS·m−1; (3) salinity treatment EC 5.5 dS·m−1, and the sub-plots included the different levels of MT treatments associated with salinity levels at EC 4.5 and 5.5 dS·m−1 (15 plants/subgroup). In each pot, the growing medium consisted of a uniform mixture of Klassmann TS3 Baltic peat (constituents are shown in Table S1), supplemented with 3 kg/m3 Osmocote Exact Potassium Dominant (Scotts, NSW, Australia) and 1 kg/m3 (soluble carbonate).

#### *2.2. Salinity Treatment and MT Application*

Prior to salinity treatments, the ranunculus seedlings were grown in separate pots for four weeks. The salt solution preparation involved adding NaCl and CaCl2 dihydrate at a molar ratio of 2:1 to attain electrical conductivity (EC) values of 4.5 and 5.5 dS·m−1 [17]; the electrical conductivity of the solution was determined using a handheld EC meter (Milwaukee EC 60 Inc, Szeged, Hungary). After six weeks of culture and salt treatments, different concentrations (0, 50, 100, and 200 μM in tap water (ethanol/water (*v*/*v*) = 1/10,000)) [38,39] of exogenous melatonin (Thermo Fischer, Geel, Belgium) were sprayed four times in two-week intervals using a handgun sprayer. The leaves were sprayed until complete wetness and dripping. Tap water was used for regular irrigation.

#### *2.3. Morphological Characteristics*

Plants (10 individual plants per treatment) were sampled two weeks after the fourth foliar application to estimate the growth parameters, such as the plant height (cm), (measured from the medium surface to the shoot apex using a meter rod), the number of leaves, (counted manually), fresh and dry weights (shoots and leaves in g). The total leaf area (Area Meter 350, ADC Bioscietific Ltd., Hoddesdon, UK) and the number of flower buds that appeared were also recorded for analysis.

#### *2.4. Physiological and Biochemical Assesments*

The five uppermost young fully expanded leaf samples were instantly frozen in liquid nitrogen and kept at −80 ◦C pending inspection. The photosynthetic pigments were measured for the acetone (80%) extract samples of each treatment and the absorbances (644, 663, and 480 nm) were recorded in a UV-VIS Spectrophotometer (Genesys 10S, Waltham, MA, USA). The carotenoid content was computed following the method of Lichtenthaler et al. [40], and the relative water content (RWC) was determined using the methods described by Turk and Erdal [41] and the formula RWC = (FW-DW)/(TW-DW) × 100.

The leaf's proline content (PC) was determined by the methods of Bates et al. [42] at 520 nm. A standard curve was constructed to calculate the proline content in μmol g−1 leaf FW. The extent of electrolyte leakage (EL) was assessed via the methods of Reddy et al. [43]. The peroxidase enzyme activity (POD) was also determined spectrophotometrically (470 nm) using the standard guaiacol method [44].

Sodium content of the leaves was determined with a flame photometer according to Campbell's method [45] and is expressed as grams per 100 g dry weight (g 100 g−1 DW). The Salt Tolerance Index (STI) was determined as a percentage (%) for each of the analyzed traits, as proposed by Sbei et al. [46]. STI = (T salt/T cont.) × 100, where T salt represents the characteristic's average value recorded under the saline conditions induced by 5.5 EC, and T cont. represents the same traits' average value under control conditions.

#### *2.5. Statical Analysis*

The experiment was set up in a completely randomized design. The Two-Way MANOVA followed by UNIANOVA for the variables with Bonferroni's correction was run for all dependent variables, between factors at two levels: (1) treatments (Cont (no salinity and no MT), Salinity EC 4.5 and Salinity EC 5.5), (2) melatonin concentrations (0, 50, 100, 200 μM). Assumptions were as if the normality of the residuals for all dependent variables are accepted by Kolmogorov–Smirnov's (*p* > 0.05) [47]. The homogeneity of variances by

Leven's F test was satisfied with all dependent variables *p* > 0.05 [48]. Tukey's post hoc test was used for factor level comparisons [49,50], while Dunnett's test was used to compare the control treatment with two salinity groups. Pairwise within-subject effects were compared via Bonferroni's method. All statistics were gathered using the software IBM SPSS27 [51].

#### **3. Results**

Statistical analysis of the obtained data revealed highly significant differences amongst multivariate factors and for the interaction effect of treatment levels (Wilk's lambda < 0.001) [52]. Thus we compared the two salinity levels with the control for the MT concentration effect, and compared the MT concentration levels with the applied salinity levels, then followed up with Univariate ANOVA for different variables using the Bonferroni's correction to see significant differences in all individual variables *p* > 0.05 [53].

#### *3.1. The Effect of Melatonin on Plant Morphology under Salinity Stress*

The applied salinity stress at both levels had an obvious negative impact on the recorded growth parameters of buttercup plants (Table 1 and Figure 1).  However, foliar melatonin treatments (50, 100, and 200 μM) considerably enhanced the plant development and improved all vegetative parameters under salinity stress. Plants treated with 200 μM MT showed the best performance under both level of salinity treatments, manifested in the remarkable increase in shoot length (23.37 and 30.04%), leaf numbers (28.32 and 21.14%), total leaf area (58.01 and 58.79%), shoot FW (29.92 and 42.33%) and shoot DW (45.20 and 41.82%), compared with stressed plants of EC 4.5 MT0 and EC 5.5 MT0, respectively, when compared with the NO-MT treated plants (Table 2).

**Table 1.** The effects of exogenous melatonin application on growth parameters of *R. asiaticus* under salinity stress.

Control (no stress–no melatonin), **S1**: plants under salt stress EC 4.5 dS·m−1 were sprayed with MT (0 μM, 50 μM, 100 μM, and 200 μM); **S2**: plants under salt stress EC 5.5 dS·m−1 were sprayed with MT (0 μM, 50 μM, 100 μM, and 200 μM); different letters are for significantly different groups (Tukey/Dunnett *p* < 0.05). The lowercase letters are for significant differences amongst melatonin concentrations under fixed salinity treatments, and the uppercase letters for significant differences in the salinity treatments with the control group under fixed melatonin concentration levels (*n* ≥ 10).

**Figure 1.** The effects of exogenous melatonin application (0, 50, 100, and 200 μM) on Na+ level (**A**), RWC (**B**), chlorophyll (**C**), and carotenoids content (**D**) of *R. asiaticus* under saline conditions. Control: no-stress and no melatonin; S1: plants under salt stress EC 4.5 dS·m−1; S2: plants under salt stress EC 5.5 dS·m−1; different letters are for significantly different groups (Tukey/Dunnett *p* < 0.05). The lowercase letters are for significant differences amongst melatonin concentrations under fixed salinity treatments, and the uppercase letters for significant differences in the salinity treatments with the control group under fixed melatonin concentration levels (*n* ≥ 5).

**Table 2.** Salt tolerance index (STI)% of *R. asiaticus* traits.

The values represent the mean of at least five replicates for the comparison of tolerance index of all estimated traits under salt stress induced by EC 5.5 dS·m−1 without melatonin application.

#### *3.2. Changes in Sodium Ion Level and Relative Water Content (RWC) of the Leaves*

Our results indicate the sensitivity of ranunculus to salinity with increased Na+. The Na+ concentration in dry leaves increased by 280 and 311% upon 4.5 (S1) and 5.5 (S2) dS·m−1 EC of irrigation water, respectively, when compared to control plants (Figure 1A). The application of exogenous MT at different concentrations (50, 100, and 150 μM) resulted in a marked reduction in Na+ content in leaf samples of *R. asiaticus* plants under both salinity levels (Figure 1A). The foliar application of 200 MT at both S1 and S2 levels revealed a significant effect and reduced the Na+ content by 50.4 and 20.5% in comparison to salinity-stressed plants without MT application (S10MT and S20MT, respectively).

The relative water content (RWC) of a plant tissue is one of the most reliable indicators of its water status and capacity for survival under stressful conditions. Our data revealed that saline conditions negatively affected the leaves' RWC, particularly at EC 5.5 dS·m−1 which had the most severe effect (Figure 1B). Compared with plants under normal conditions (non-stressed plants), stressed plants, S1 0MT and S2 0MT exhibited a significant decrease (21.1 and 28.3%, respectively) in RWC.

On the other hand, the foliar melatonin treatment showed a slight improvement in RWC under the saline conditions. The increase in RWC was noted in the presence of 100 and 200 μM melatonin compared with non-MT-treated stressed plants which was still lower than that of control plants (Figure 1B). These outcomes imply that the foliar melatonin treatment has more favorable impacts on plant biomass and development upon salt stress, and that the 200 μM melatonin could better protect the plants under saline conditions than 100 and 50 μM melatonin.

#### *3.3. Changes in Photosynthetic Pigments of Leaves*

Saline conditions negatively affected the leaves' total chlorophyll (Chl) and carotenoid (Car) content of buttercup plants, with the influence being most prominent at the higher NaCl concentration of EC 5.5 treatment (Figure 1C,D). The leaves' Chl and Car content of non-MT-treated plants under both salinity levels (S1 0MT and S2 0MT) were estimated for a decrease of 45.12 and 55.49% in cases of chlorophyll and 17.24, and 44.82% in cases of carotenoids, respectively. However, the exogenous MT application at different doses (50, 100, and 150 μM) on *R. asiaticus* plants resulted in a significant increase in leaves' photosynthetic pigments (total Chl and Car) in comparison to stressed plants without MT under both salinity levels (Figure 1C,D). The foliar application of 200 MT at both S1 and S2 levels revealed a significant effect, with the rise of leaves' Chl and Car estimated at 68.89 and 83.56%, respectively, compared with stressed plants without MT application (S10MT and S20MT, respectively). These data indicate that 200 μM MT was able to inhibit the entrance of detrimental ions into the cells, safeguarding the cellular structure.

#### *3.4. Changes in Electrolyte Leakage and Proline Content*

The impacts of salinity stress and exogenous melatonin application on membrane integrity and electrolyte leakage (EL) are illustrated in Figure 2A. Saline conditions induced by NaCl 4.5 and 5.5 dS·m−1 significantly increased the electrolyte leakage by 64.01 and 67.74%, respectively, when compared to control plants. In contrast, in stressed plants subjected to foliar application of melatonin (50 μM, 100 μM, and 200 μM), there was a significant and concentration-dependent decrease in electrolyte leakage. The application of 200 μM MT resulted in the highest reduction of EL by 28.93 and 20.57% under both S1 and S2 salinity levels, when compared with salt-stressed plants without MT treatment (Figure 2A).

**Figure 2.** The effects of exogenous melatonin application (0, 50, 100, and 200 μM) on electrolyte leakage (**A**), proline content (**B**), peroxidase enzymes activity (**C**), and flower bud emergence time (**D**) of *R. asiaticus* under saline conditions. Control: no stress and no melatonin; S1: plants under salt stress EC 4.5 dS·m−1; S2: plants under salt stress EC 5.5 dS·m−1 different letters are for significantly different groups (Tukey/Dunnett *p* < 0.05). The lowercase letters are for significant differences amongst melatonin concentrations under fixed salinity treatments, while the uppercase letters are for significant differences in the salinity treatments with the control group under fixed melatonin concentration levels (*n* ≥ 5).

Proline, a compatible membrane protective solute with its role in maintaining water balance and enhancing cytoplasmic osmotic pressure is known to protect the cells during early dehydration. According to our findings, saline conditions showed a considerable rise in proline content in leaves, with its highest values being recorded at the S2 (EC 5.5) treatment. Stressed plants (S1 0MT and S2 0MT) exhibited a significant increase of about 50% in proline content in comparison with control plants. The foliar melatonin application at 50, 100, and 200 μM resulted in a gradual increase in proline content under the saline conditions when compared to non-MT-stressed plants (Figure 2B).

#### *3.5. Changes in Peroxidase Enzyme Activity (POD)*

A significant increase (*p* < 0.05) in POD activity was detected in buttercup plants under both salinity stress conditions (Figure 2C). The POD levels increased by 61.9 and 82.2% when plants were irrigated with saline water with EC of 4.5 and 5.5 dS·m−1, respectively, when compared to non-stressed plants. Under both S1 and S1 salinity conditions, all of the melatonin treatments (50 μM, 100 μM, and 200 μM) significantly increased (*p* < 0.05) the POD activity, with its highest level being detected after 200 μM MT treatment. A 48.8 and 46.7% increase in antioxidant enzyme activity was recorded in plants under S1 and S2

conditions, respectively, when treated with 200 μM MT compared with non-MT-stressed plants under both salinity levels. These findings suggest that the NaCl stress brought the POD activities to a certain level as expected and that the foliar MT application enhanced (in a linear concentration-dependent manner) the POD activity even more to protect the cells from ROS accumulation.

#### *3.6. Changes in Flower Bud Emergence*

Our results reveal that both salt stress conditions (S1 and S2) induced the flower buds' appearance (Figure 3). Flowering time of salt stressed plants irrigated with saline water with EC of 4.5 and 5.5 dS·m−1 without melatonin treatment occurred 29 and 36 days sooner than in the control plant, respectively. This early flowering time was delayed after the application of melatonin for 22, 24, and 15 days in the case of S1 treatment and 17, 8, and 10 days in the case of S2 salinity treatment when 50, 100, and 200 μM of MT was applied, respectively, in comparison to control plants (unstressed plants without MT).

**Figure 3.** Morphological changes in *R. asiaticus* plants being treated with different concentrations of exogenous melatonin under salinity stress.

#### *3.7. Salt Tolerance Index (STI)*

Table 2 depicts the STI as a percentage of all examined variables between the nonmelatonin-treated plants grown under unstressed conditions and stressed plants under S2 salinity level (EC 5.5 dS·m−1). The STI% demonstrated that Na+ content in leaves was the trait most sensitive to saline conditions, compared to STI% of other variables, which gave 437.9%, followed by POD activity and El value in the second place, which gave 321.3 and 309.9%, respectively. Proline content was in the third place for the salt response, which gave 202.5%. In addition, without significant differences, the remaining traits exhibited STI% values less than 100% and higher than 50%, except total Chl showed the lowest salinity-responsiveness, which gave 44.4%.

#### **4. Discussion**

Salinity stress is known to cause plant performance to deteriorate due to osmotic, ionic, or nutritional disorders, represented in the morphological characteristics of subjected plants. As an activator of antioxidant enzymes, melatonin is known to protect plants from oxidative stress [54]. The cut flower species and in particular the ranunculus is reported to be sensitive to salinity stress [17,18]. In the present work, exposing ranunculus plants to salinity stress induced by EC 4.5 and EC 5.5 caused a significant reduction in all the assessed vegetative growth characteristics when compared with unstressed plants (control) where the EC 5.5 was more harmful (Table 1 and Figure 1). Similarly, it has been reported that increasing EC from 2 to 3 dS·m−1 significantly stunted plant growth, dry weight, number of leaves, and diameter of flowering stems of white and pink *R. asiaticus* cultivars [18]. Poorter et al. [55] reported that the plant leaf-related parameters were significantly affected by salinity.

Melatonin (MT) is known as an endogenous regulator with an enhancement effect on both plant growth promotion [36,56,57] and also in protecting against abiotic stress [31,58–61] even when applied in small concentrations [62]. Many investigations have demonstrated MT's efficacy as a dose-dependent stimulator of plant growth and development [35,63,64]. We evaluated the effects of melatonin on ranunculus plants under saline conditions and found that foliar spray with 200 μM MT was the most effective concentration to mitigate the adverse effects of stress in most of the traits studied. Our findings were in line with the recent investigation on cotton seedlings treated with 200 μM of melatonin [65]. Exogenous melatonin must be applied to plants at the appropriate concentration, which may be different for distinct plant species [66]. Melatonin. an indoleamine, shares IAA's metabolic precursor, which may explain its influence on plant growth and development [67]. In addition, earlier research reported that environmental stress factors may induce flowering to be able to perpetuate the species' survival [68,69]. Exogenous melatonin was involved in flowering bud growth, as reported by Kolár et al. [ ˇ 70]. Similar to our results (Figure 2D), in Arabidopsis, exogenous MT caused a delay in flowering time [71]. This may occur due to the involvement of exogenous MT in upregulation of flowering locus C (FLC), which suppresses the transcription of flowering locus T (*FT*) [56,72].

Excessive cytoplasmic NaCl levels interrupt ion balance and inhibit plant growth and development [73]. Accordingly, plants must be able to ingest and compartmentalize ions to increase their salt tolerance efficiently. Thus, plants redistribute cytoplasmic salt ions into vacuoles or store them in various tissues to cope with salinity stress [74,75]. Consequently, a more significant Na+ build-up will be observed in leaves. Likewise, in this experiment, Na+ content of the leaves dramatically increased at both saline conditions EC 4.5 and 5.5 dS·m−1 (Figure 1A). Similar outcomes were also noticed in the cotton leaves [65] and pepper cultivars 'Granada' and 'Nobili' [76]. However, exogenous MT treatment allowed ranunculus plants to retain lower Na+ in leaves than in non-MT-treated stressed plants (Figure 1A), and higher reduction was observed under S1 conditions by increasing MT levels. These findings agree with Castañares and Bouzo [36] and Wei et al. [77], reporting that MT treatment reduced ion toxicity in melons and rice, respectively when subjected to salt stress. This research also confirmed that 200 μM MT aids in preserving ion equilibrium while exposed to salt stress. Specifically, melatonin's action in the presence of saline stress is to upregulate the transporter genes *NHX1* and *AKT1* [78], which are involved in preserving the ion homeostasis. Due to the extreme sensitivity of plants to salinity, water status is the primary factor in response to stress [79]. Using water relation properties in ranunculus leaves, we found a common reactivity to salinity, consistent with the findings of St ˛epie ´n and Kłobus [80] on cucumber and on pepper cultivars [76]. Toxic ion accumulation, especially Na+ and Cl−, may be to blame for the reduction in RWC by causing leaves to shrink and their stomata to close, thereby lowering the intracellular CO2 partial pressure [81]. According to Munns' [82] study, higher sodium chloride concentrations led to a greater loss of water from the cells, which manifested as dehydration; thus, the cell growth rate was rapidly reduced in addition to a series of metabolic alterations like those imposed by

water stress. MT application in our study, however, increased RWC gradually by increasing MT concentrations and the higher concentration was the most effective under both salinity levels S1 and S2 (Figure 1B) similar to those reported in borage plants [79], likely attributable to enhanced water absorption due to safeguarding the cell membrane [36,80]. It has been proven that melatonin can improve the thickness of a plant's cuticle, which in turn helps to limit water loss. Melatonin treatment has been shown to improve stress tolerance in plants by keeping their turgor and water ratio stable [81].

Photosynthesis relies on chloroplast chlorophyll, which can capture and transmit light energy for plants and is a key indicator of plant physiological properties and performance [82]. In plants subjected to saline conditions, a decrease in total chlorophyll and carotenoids was observed, and a more severe effect was found under S2 conditions (Figure 1C,D). This is correlated with excessive quantities of Na+ that result in sluggish synthesis or rapid breakdown of photosynthetic pigments, which has an immediate impact on plant growth [83]. An application of melatonin in earlier studies enabled salt-stressed plants to keep their chlorophyll levels closer to normal such as in melon (*Cucumis melo* L.) [36], pistachio [60], maize seedlings [29], and in spearmint plants [84]. Similarly, in the current study, exogenous melatonin enhanced chlorophyll and carotenoid levels in ranunculus plants compared to non-MT-stressed plants. In general, S1 (4.5 EC) showed the higher pigment accumulation with MT application, and 200 μM MT was the most effective concentration applied under both salinity stress levels, supporting the hypothesis that MT pretreatment promotes biosynthesis and reduces chlorophyll breakdown [85]. This effect has been attributed to melatonin's antioxidant properties and its influence on the genes for the chlorophyll-degrading enzymes CLH (chlorophyllase), PAO (pheophorbide an oxygenase), and the red chlorophyll catabolite reductase [56]. Additionally, high melatonin dosages improved chloroplast carotenoid content and lipid-soluble antioxidants [74]. Consequently, melatonin can stimulate carotenoid production and result in lowering the levels of photooxidative degradation [31].

Ion-specific salt degradation mainly occurs in the plasma membrane [86]. Electrolyte leakage can be attributed to a loss of membrane integrity, which reduces plants' ability to hold K+ as a result of stress [87]. Consequently, plasma membrane electrolyte leakage is regarded as an essential indicator for selecting tolerant plants under salt stress [86]. From our data, both salinity levels increased EL rate, but S2 (EC 5.5) caused the highest EL rate. However, minimizing EL in plants treated with MT afforded a protective effect toward membrane damage under saline conditions (Figure 2A). Similar results were presented by Khan et al. [88] and Zhang et al. [89], who reported that MT treatment reduced EL in sugar beets and tomato under salinity stress, indicating that MT may alleviate the oxidative damage induced by salt conditions. Moreover, melatonin, is present on the hydrophilic side of lipid bilayers and can diffuse through lipid membranes and the cytoplasm [90–92]. Melatonin's ability to organize itself in lipid membranes is concentration-dependent as at lower doses the molecules align themselves parallel to the lipid tail, while at higher doses they arrange themselves parallel to the bilayer [92].

Plants accumulate osmotic regulators as part of their adoptive stress-reduction strategy to preserve intracellular stability and shield their cells from the detrimental consequences of saline stress [64,93]. Ferchichi et al. [94] revealed that proline serves numerous functions, including stability of membranes and proteins, regulating gene expression in response to salinity conditions to maintain the redox balance. Previous investigations have shown that the melatonin-induced buildup of certain osmolytes diminishes cells' osmotic potential, which enhances osmotic adjustment and hence increases plants' water content under stressful environments possibly by upregulating the transcription of genes like *BADH* and *P5CS* [36,89]. Melatonin stimulates root development and aquaporin function, which in turn stimulate water uptake and distribution, and ultimately improves hydration status under salt conditions [95]. This mechanism is supported by the findings of the current study (Figure 2B), when MT enhanced proline accumulation under both salinity levels compared to non-stressed plants. Likewise, Sheikhalipour et al. [64] observed that MT-treated plants

showed increased RWC and proline content under salinity conditions when compared with non-MT-treated plants under stress.

Additionally, ROS is a significant indicator of oxidative and salt stress [96]. The antioxidant machinery present in cells is responsible for maintaining ROS homeostasis, which is necessary for cells to avoid irreparable damage and maintain their integrity (antioxidant compounds and enzymes) [77,97]. The primary function of peroxidases as an ROS scavenger is to convert H2O2 into water molecules after SOD has converted O2 •− to O2 and H2O2 [98]. Exogenous MT decreased the formation of ROS by scavenging ROS and promoting antioxidant activity [99]. In this investigation, MT treatments, especially those with a concentration of 200 μM MT (Figure 2D), enhanced the POD activity under stressful conditions and the highest activity was noticed under the S2 treatment (EC 5.5). Our results also corroborate the findings of Zhang [28] that MT can improve salt tolerance by upregulating antioxidant enzyme genes and lowering biological macromolecule breakdown to increase antioxidant enzyme activity under high salinity. Similar investigations into rice [77], naked oat [100], and rapeseed [101] showed the same trend.

Depending on the salinity tolerance index (STI%) values under severe stress of EC 5.5 (S1 treatment), the investigated characteristics in this study could be divided into three categories (Table 2). The first category consists of the variables having an STI% greater than 400%, as observed with Na content; the second category is the variables that exhibited STI% of more than 200%, as observed with POD activity, El, and proline. While the final category includes the variables with STI% values below 100%, as observed with flower bud emergence time, RWC, number of leaves, shoot length, shoot fresh weight, carotenoids, shoot dry weight, total chlorophyll, and leaf area, in descending order. Based on the high STI% values for Na content, followed by POD activity, El, and proline, it is conceivable to use these characteristics as obvious markers of ranunculus plants' response to salt stress. Some scientific papers have debated the fractionated STI as a stress-response indicator and considered rather the biomass of the entire plant, for instance, as reported for chickpea [102] and Asian barley [46]. In this work, it can be stated that STI% was computed in a wide variety of ranunculus plant features, similar to an approach by Roshdy et al. in strawberry plants [103], that are more informative for future studies and plant breeding projects.

#### **5. Conclusions**

This study investigated the effect of exogenous melatonin treatment on *R. asiaticus* under two salinity levels (EC 4.5 and EC 5.5). The melatonin application enhanced RWC content and photosynthesis pigments, and reduced Na+ accumulation in leaves, resulting in strengthening the vegetative and growth parameters under saline conditions in a dosedependent manner. Additionally, our data showed improvement in osmotic regulation capability by increasing osmolyte accumulation (Proline), as well as the protective evidence that exogenous melatonin application in buttercup seedlings enhanced the performance of the antioxidant defense system by diminishing the ROS generation as demonstrated by activation of POD and the decrease in EL. Considering STI% values under the most harmful salinity level (EC 5.5), sodium content in leaves, followed by enzyme activity, EL, and leaf proline content could indicate a Ranunculus plant's salinity stress response. Overall, when applied at the optimal dose of 200 μM in this study, melatonin alleviates salinity stress on morpho-physiological characteristics, improving *R. asiaticus*' tolerance, which can be considered as an effective practice for productions under stress conditions.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/horticulturae9020228/s1, Table S1: Klassmann TS3 Baltic peat chemical components.

**Author Contributions:** Conceptualization, A.T.-M. and P.H.; methodology, E.A.E., A.T.-M. and P.H.; validation, E.A.E., A.T.-M. and I.M.; formal analysis, E.A.E.; investigation, E.A.E.; resources, A.T.-M.; data curation, E.A.E. and I.M.; writing—original draft preparation, E.A.E.; writing—review and

editing, I.M.; visualization, E.A.E. and I.M.; supervision, A.T.-M. and P.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no funding. The APC was paid by the Hungarian University of Agriculture and Life Sciences (MATE).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** E.A.E. is thankful to the Tempus Public Foundation of Hungary for the Stipendium Hungaricum scholarship.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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## *Article* **The Effective Role of Nano-Silicon Application in Improving the Productivity and Quality of Grafted Tomato Grown under Salinity Stress**

**Eman G. Sayed 1,\*, Abdel Wahab M. Mahmoud 2, Mohamed M. El-Mogy 1, Mahmoud A. A. Ali 3, Mahmoud A. M. Fahmy 4 and Ghada A. Tawfic 1**

**Abstract:** This study aims to determine the influence of grafting and nano-silicon fertilizer on the growth and production of tomatoes (*Solanumlycopersicum* L.) under salinity conditions. A commercial tomato hybrid (cv. Strain B) was used as a scion and two tomato phenotypes were used as rootstocks: *S. pimpinellifolium* and Edkawy. The rootstock effect was evaluated by growing plants at two NaCl concentrations plus the control (0, 4000, and 8000 ppm NaCl). Nano-silicon foliar application (0.5 ppm) after 20, 28, and 36 days from transplanting was also used to mitigate salinity stress. Antioxidants, hormones, and proline were evaluated for a better understanding of the physiological changes induced by salinity and grafting. The results showed that grafting either on *S. pimpinellifolium* or Edkawy combined with nano-silicon application enhanced shoot and root growth, fruit yield, and fruit quality. The Edkawy rootstock was more effective than the *S. pimpinellifolium* rootstock in terms of counteracting the negative effect of salinity. Higher levels of mineral contents, GA3, ABA, and proline were detected in shoots that were subjected to grafting and nano-silicon application compared to the control treatment. This study indicates that grafting and nano-silicon application hold potential as alternative techniques to mitigate salt stress in commercial tomato cultivars.

**Keywords:** *Solanum lycopersicum* L.; salinity; quality; nano-silicon; plant hormones

#### **1. Introduction**

The tomato plant (*Solanum lycopersicum* L.) is nowadays a very popular crop as a means of preventing many human diseases [1,2] and its fruit is well known for carotenoids like lycopene that seem to be active in cancer prevention, cardiovascular risk, and cellular aging [3]. Tomato is one of the most common and widely consumed vegetable crops in the world, and in Egypt a high-quality yield is a must for commercial success. Salinity is one of the most important abiotic stress factors limiting crop growth and productivity worldwide [4]. High salinity leads to a decrease in plant growth, biomass, yield, photosynthesis, and water use efficiency [5], as salinity stress negatively impacts the morphological, biochemical, and physiological processes of plants [6,7]. Salt stress reduces the growth and yield of grafted and non-grafted tomato plants, but the appropriate combination of scion and rootstocks can help mitigate the negative effects of salinity. An effective and sustainable method of improving the performance of commercial cultivars that are susceptible to abiotic stresses is to use resistant genotypes as rootstocks [8]. The local Egyptian cultivar Edkawy exhibits greater salinity tolerance by exhibiting greater growth stability as salinity

**Citation:** Sayed, E.G.; Mahmoud, A.W.M.; El-Mogy, M.M.; Ali, M.A.A.; Fahmy, M.A.M.; Tawfic, G.A. The Effective Role of Nano-Silicon Application in Improving the Productivity and Quality of Grafted Tomato Grown under Salinity Stress. *Horticulturae* **2022**, *8*, 293. https://doi.org/10.3390/ horticulturae8040293

Academic Editors: Agnieszka Hanaka, Małgorzata Majewska and Barbara Hawrylak-Nowak

Received: 14 February 2022 Accepted: 28 March 2022 Published: 30 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

increases [9]. In addition, *S. pimpinellifolium* is identified as an important source of genes that can help mature tomato plants cope with stress [10]. Conversely, it has been demonstrated that salinity eustress may contribute to augmenting organoleptic components of fruit quality, such as soluble carbohydrates and health-promoting phytochemicals [6,11]. Moreover, numerous experiments have indicated the beneficial role of silicon (Si) against different stresses, including salinity in tomatoes [12]. Exogenous Si spraying has been a recent eco-friendly strategy to improving plant salinity stress response [13] as it acts by increasing plant biomass [14] by reducing Na+ and Cl− ion uptake into plants [15]. Silicon is found in nature as crystalline, amorphous, or weakly crystalline complex silicate minerals [16,17]. It also aids plant growth in a variety of ways by improving antioxidant activity, mineral absorption, organic acid anion and phenolic compound exudation, photosynthetic rates, the accumulation of suitable solutes, hydration status, and the control of plant growth regulators [18–28], as well as considerably lowering the deleterious impact of salinity on chlorophyll levels [29]. Nanotechnologies and plant biotechnology have attracted a lot of attention in agriculture in recent years [30,31] on account of their potential to increase plant productivity, improve plant tolerance to environmental stress conditions, improve nutrient use efficiency, and decrease hazardous environmental consequences [31–33]. Crop improvement experiments have been carried out with silicon nano-particles (n-Si) and many studies have found that increasing n-Si concentrations improves plant development and tolerance to hydroponic conditions [34]. Tomato plants treated with 1 mM n-Si displayed increased germination rates and seedling dry weight, thereby showing higher salinity tolerance at 50 mM NaCl compared with the controls [35,36]. A similar effect was shown in lentil seeds [37]. Nano-silicon sprays increased the amount of chlorophyll in stressed plants and enhanced physiological parameters, such as transpiration rate, photosynthetic rate, stomatal conductance, and photochemical efficiency, in *Indocalamus barbatus* [38]. Moreover, (n-Si) enhanced the nutritional quality of potatoes that had been exposed to salt [39], and n-Si spray significantly improved plant height, stem diameter, ground cover, canopy spread, and other growth characteristics in safflower [40]. Based on this body of evidence, the purpose of this study is to assess whether grafting and foliar application of n-Si have a positive effect in terms of improving the yield and quality of a tomato hybrid by evaluating plant growth characteristics, yield, and fruit quality traits.

#### **2. Materials and Methods**

#### *2.1. Plant Material*

In this study, the hybrid cv. Strain B tomato obtained from Ferry-Morse seed CO USA was employed as a scion. The seeds of a tomato rootstock with documented salinity tolerance features, *S. pimpinellifolium* L. (line AusTRCF31212), were obtained from the International Tomato Genetic Resource Center in the United States. In addition, Edkawy (obtained from Haraz company, Cairo, Egypt) was used in this study as a local cultivar with recognized tolerance qualities.

#### *2.2. Grafting of Test Plants*

Seeds of the scion and rootstock were sown on 21 March 2020 and 17 March 2021 in seedling trays filled with peat moss and vermiculite (1:1 *v:v*). Scion seedlings were grafted on rootstock manually via tongue grafting on 20 and 15 April in 2020 and 2021, respectively. Both rootstock and scion grafts were sliced obliquely at a 40◦ angle to the perpendicular axis at a sufficient depth to allow for greater vascular bundle overlap. Clips were applied at the grafting site to secure the graft in place, and the grafted plants were maintained under a clear polyethylene tunnel cover for 5–7 days under 90–95% RH and 50% shading at a temperature of 27.9 ◦C. The polyethylene cover was gradually opened to acclimatize the grafted plants to the greenhouse environment. Three sets of grafted plants were produced using the commercial tomato hybrid (cv. Strain B) as a scion grafted on one of two tomato rootstocks, namely, Edkawy and *S. pimpinellifolium*, respectively, or on the same hybrid (cv. Strain B) to serve as a basis for the control treatment.

#### *2.3. Greenhouse Experiment*

#### 2.3.1. Growth Conditions

Transplantation of grafted plants was carried out in the greenhouse at the Eastern Farm of the Faculty of Agriculture, Cairo University, Giza, Egypt on 28 April in 2020 and on 23 April in 2021. The three groups of successfully grafted tomato plants were transplanted in pots 60 cm2 in diameter, each one filled with a 1:1:1 mixture of peat moss, vermiculite, and perlite and containing two seedlings, and were each subjected to (i) three salinity levels—0 ppm, 4000 ppm, and 8000 ppm of NaCl solution—and (ii) treatment with or without nano-silicon (n-Si) foliar application (0.5 ppm) three times after 20 and 28 and 36 days from transplanting. The reported design resulted in 18 treatment combinations. Physiological and biochemical investigations were performed 60 days after transplantation.

#### 2.3.2. Salinity Treatments

Thirty days after transplantation, the NaCl solution was applied to the levels of saline treatments, which contained 0, 4000, and 8000 ppm NaCl (Technogene chemical company, Dokki, Egypt). The saline treatments were continued until the experiment was completed (190 days after seedling transplantation). A completely randomized design was used for the treatments and each treatment was replicated six times.

#### 2.3.3. Preparation of Nano-Silicon

Nano-silicon in the form of silicon tetrachloride (SiCl4) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The nano-silicon (Figure 1) was synthesized using the method described by Zhu and Gong [41] and published elsewhere [42]. The morphologies and sizes of the nano-particles (n-Si) were investigated using a JEOL 1010 transmission electron microscope at 80 kV (JEOL, Tokyo, Japan). One drop of the nano-particle solution was spread onto a carbon-coated copper grid for transmission electron microscopy (TEM) analysis. TEM imaging was carried out in the TEM lab of the Faculty of Agriculture, Cairo University Research Park (FA-CURP) to determine the nano-size of the silicon particles.

**Figure 1.** Scanning electron microscopy (SEM) for the prepared silicon nano-particles. Silicon nano-particle size from 4.75 to 6.92 nm.

#### 2.3.4. Plant Growth and Yield

Plant growth was measured as a function of shoot length, plant fresh weight, leaf number, and leaf dry weight. Leaf chlorophyll content was recorded using a SPAD Meter. The fruits of each plot were picked during harvesting, and the number of fruits per plant, fruit fresh weight, and total yield per plant were recorded.

#### 2.3.5. Characteristics of Fruit Quality

At the maturity stage, ten mature fruits per experimental unit were chosen to measure the following data. Total soluble solids percentage (TSS%) was measured using a Zeiss laboratory refractometer, and the ascorbic acid (AA) content of ripe fruits was evaluated via the titration method using 2,6-dichlorophenol indophenol according to the Society for Analytical Chemistry's Official Method 967.21. Five fruits were selected in each replicate to determine their firmness. The firmness of ripe fruits was determined using a Force Gauge Mode M4-200 (ELECTROMATIC Equipment Co., Inc., Cedarhurst, NY, USA) with a 1 mm diameter flat probe. The firmness readings of tomato fruits were taken at two opposing points of the equatorial region and expressed in newtons.

#### 2.3.6. Physiological Parameters

To evaluate photosynthesis and leaf stomatal conductance, analysis was conducted via an infrared gas analyzer, the LICOR 6400 Portable Photosynthesis System (IRGA, Licor Inc., Lincoln, NE, USA), on the fifth leaves of twenty plants chosen from each treatment with six replications. Measurements were taken from 9 a.m. to 2 p.m., with a light intensity of around 1300 mol m−2 s−1 and 80% RH. The leaf chamber's temperature ranged from 25.2 to 27.9 ◦C, and the volume gas flow rate was 400 mL/min. The CO2 content in the air was 398 μmol mol−1.

#### 2.3.7. Activity of Antioxidant Enzymes, Gibberellic Acid, Abscisic Acid Content, and Leaf Proline

The leaf samples used to measure the activity of peroxidase (POD) were prepared according to Bates et al. [43]. The free proline content was analyzed as described by Bates et al. [43]. After homogenisation, 500 mg of freeze-dried materials in 5 mL of sulphosalicylic acid at 3% (*w/v*) was filtered using Whatman No. 1 filter paper. The filtrate was combined with acetic acid and a ninhydrin acid reagent (2% *v*/*v*). Then, for 45 min, the mixture was immersed in a boiling water bath at 100 ◦C. The tubes were then filled with 4 mL of toluene and soaked for 20 s, after which the reaction was quenched by submerging the tubes in an ice-bath. The free proline was measured spectrophotometrically against a blank reagent at 520 nm. The contents of gibberellic acid (GA3) and abscisic acid (ABA) in tomato leaves were analyzed using the method described by Fales et al. [44]. A 15 mL aliquot of a methanol/butylated hydroxytoluene (80% *v*/*v*) solution was mixed with the homogenized freeze-dried samples, and GA3 and ABA were extracted and quantified according to AOAC guidelines [45].

#### 2.3.8. Mineral Composition in Tomato Plants

The tomato leaf samples were dried for two days at 75 ◦C in a forced air oven and then coarsely pulverized for the determination of endogenous nutrients. Sulfuric acid (5 mL) and perchloric acid were used to digest a 0.2 g dried sample. The mixture was then heated for ten minutes and 0.5 mL of perchloric acid was added while continuing to heat the mixture until clear [46,47]. The total nitrogen (N) content of the dried leaf samples was analyzed using the AOAC-recommended modified micro-Kjeldahl method described by Singh et al. [46] The phosphorus (P) content was determined colorimetrically using the chlorostannous molybdophosphoric blue color method in sulfuric acid, according to [46]. A flame photometer (CORNINGM410, Halstead, UK) was used to test the content of potassium (K), magnesium (Mg), calcium (Ca), and sodium (Na). An atomic absorption spectrophotometer with air-acetylene fuel (PyeUnicam Company, model SP-1900, Ventura, CA, USA) was used to examine the iron (Fe) and zinc (Zn) concentrations.

#### 2.3.9. Statistical Analysis

A randomized complete block design with two factors was used to analyze all the data obtained from six replicates during two growing seasons using the computer application "MSTATC" [48]. The LSD test was used to assess changes across treatment modalities at a 5% probability level [49].

#### **3. Results**

#### *3.1. Plant Growth Parameters*

Plant growth parameters were affected by salinity levels and grafting (Table 1). The results of the present experiment indicated that among the tested water salinity concentrations, plant growth parameters decreased with increasing salinity.

**Table 1.** Effects of water salinity, grafting, foliar application of nano-silicon (n-Si), and their interactions on the vegetative growth of tomato plants (combined 2020 and 2021 seasons).

Values followed by the same letter are not significant according to the LSD test (*p* ≤ 0.05%).

In general, it was observed that plant height was inversely proportional to salinity concentration. In the Edkawy cultivar, the n-Si treatment had a positive influence on plant height under no salinity (248.0 cm with 0 ppm) or moderate stress (213.7 cm with 4000 ppm). The leaf count per plant was slightly affected by moderate salinity and the results were comparable to those obtained in the no-salinity treatment (31.3 with 4000 ppm, 35.3 with 0 ppm) with interaction between grafting and nano-silicon (Table 1). Additionally, in the Edkawy rootstock, it was observed that the fresh weights of shoots and roots were higher when the grafting and n-Si treatments were combined. A slight reduction in fresh root weight was observed (70.60 g) under 4000 ppm salinity and n-Si treatment when compared with the control of the same rootstock (81 g) with unstressed plants without nano-silicon application; a similar trend was observed for the dry weight parameter. As can be seen, plant growth parameters decreased with increasing salinity levels, but the combination of grafting and n-Si foliar application mitigated the effects of salinity (Table 1).

#### *3.2. Tomato Fruit Yield and Its Components*

Fruit number and fruit weight as well as total fruit yield per plant were significantly reduced with increasing salt levels. The self-grafted Strain B hybrid was the most negatively affected, particularly at 8000 ppm concentration. Grafted tomato plants subjected to the n-Si treatment registered greater fruit numbers. Similarly, significantly higher fruit number, fruit weight, and total yield per plant values were recorded for the Edkawy rootstock combined with n-Si treatment when compared with the untreated control under all three salinity levels. Notably, the n-Si treated Edkawy at 4000 ppm and 8000 ppm yielded fruit numbers comparable to the untreated Edkawy plants at 0 ppm (Figure 2A).

**Figure 2.** Effects of water salinity levels × rootstocks and foliar spray with (n-Si) interaction on (**A**) average number of fruits, (**B**) average fruit weight (g), and (**C**) average total yield per plant (kg). Vertical bars represent standard errors of the mean; in each bar, values followed by different letters differ significantly at *p* = 0.05 according to the LSD test.

The highest mean fruit weight was recorded using Edkawy combined with nanosilicon under 0 and 4000 ppm of salt concentration (Figure 2B). Plants grafted and combined with n-Si application had higher yields per plant (Figure 2C), although, at 4000 and 8000 ppm, all treatments had a higher value than self-grafted plants. Interaction between Edkawy and n-Si showed higher fruit weight at 0 ppm. Plants grafted on Edkawy and combined with nano-silicon had higher total yields per plant under non-saline water treatment.

#### *3.3. Physiological Traits of Tomato Plants*

Transpiration rate, stomatal conductance, photosynthesis, and chlorophyll reading (SPAD) were significantly influenced by salinity levels, grafting, foliar application of n-Si, and the interactions between them (Figure 3). The values obtained for the physiological traits were higher at 0 ppm salinity; however, no statistically significant differences were observed between n-Si and untreated counterparts of the same cultivars. Overall, all grafting combinations, with the exception of the self-grafted Strain B hybrid, showed similar levels of transpiration rates, stomatal conductance, photosynthesis, and SPAD readings, with only minor statistically significant differences. In this case, also, the self-grafted Strain B hybrid had the lowest transpiration rate, stomatal conductance, photosynthesis, and SPAD reading, and, notably, the negative impact of salinity was reduced for its grafted counterpart. Edkawy with nano-silicon and *S. pimpinellifolium* with nano-silicon exhibited better stomatal conductance and photosynthesis under 4000 ppm levels. SPAD showed no significant change between the control and 4000 ppm using Edkawy combined with the nano-silicon application.

**Figure 3.** *Cont*.

**Figure 3.** Effects of water salinity levels, rootstocks, foliar spray with (n-Si), and their interactions on (**A**) transpiration rates, (**B**) stomatal conductance, (**C**) photosynthesis and (**D**) SPAD readings. Vertical bars represent standard errors of the mean; in each bar, values followed by different letters differ significantly at *p* = 0.05 according to the LSD test.

#### *3.4. Quality Parameters of Tomato Fruits*

Total soluble solids (TSS), firmness, and vitamin C values for tomato fruits were significantly influenced by salinity levels, grafting, foliar application of nano-silicon, and their interactions (Table 2). TSS, firmness, and vitamin C values for tomato fruits were increased considerably as a result of increasing salinity. Tomato fruits from the Edkawy rootstock with nano-silicon and from the *S. pimpinellifolium* rootstock with n-Si showed significantly greater TSS percentages compared to other treatments. Harvested fruits from the self-grafted Strain B hybrid had lower quality parameters than fruits from tomato plants subjected to the 8000 ppm concentration treatment.

**Table 2.** Effect of water salinity, grafting, foliar application of nano-silicon (n-Si), and their interactions on the quality parameters of tomato fruits (combined 2020 and 2021 seasons).

Values followed by the same letter are not significant according to the LSD test *(p* ≤ 0.05%).

#### *3.5. Mineral Content in Tomato Shoots*

Measured mineral traits in tomato shoots were significantly influenced by salinity, grafting, foliar application of nano-silicon, and their interactions (Table 3). Increasing salinity concentration significantly reduced N, P, K, Ca, and Mg contents in tomato shoots. All treatments accumulated minerals more than the self-grafted strain B hybrid. At 4000 ppm levels, plants grafted and combined with foliar nano-silicon had better shoot mineral concentrations, although the self-grafted strain B hybrid showed the lowest concertation of N, P, K, Ca, and Mg under 8000 ppm levels.

Tested Na, Fe, and Zn in tomato shoots were also considerably affected by salinity, grafting, foliar application of nano-silicon, and their interactions (Table 4). Increasing salinity concentration significantly reduced Fe and Zn contents in tomato shoots, while Na increased with increasing salinity.

Edkawy with nano-silicon exhibited the lowest content of Na and the highest contents of Fe and Zn. On the other hand, the self-grafted strain B hybrid recorded the higher content of Na. Nevertheless, the self-grafted strain B hybrid had the lowest concentrations for Fe and Zn. Salinity, grafting, and foliar application of nano-silicon interaction showed various meaningful influences. The highest Na content appeared with the self-grafted strain B hybrid at 8000 ppm salt levels. Despite this, the lowest Na content appeared using a combination of grafting with nano-silicon under control. *S. pimpinellifolium* combined with nano-silicon had a high Zn content under 4000 ppm levels compared with other treatments.

**Table 3.** Effect of water salinity, grafting, and foliar application of nano-silicon (n-Si) interactions on the mineral contents of tomato shoots (combined 2020 and 2021 seasons).

Values followed by the same letter are not significant according to the LSD test (*p* ≤ 0.05%).

**Table 4.** Effect of water salinity, grafting, and foliar application of (n-Si) interactions on minerals of tomato shoots (combined 2020 and 2021 seasons).

Values followed by the same letter are not significant according to the LSD test (*p* ≤ 0.05%).

#### *3.6. Plant Hormones, Antioxidant Enzymes, and Proline Content in Tomato Shoots*

Salinity, grafting, foliar application of nano-silicon, and their interactions result in variations and changes in the activity of the most important hormones (GA3, ABA) and POD antioxidant enzymes as well as the amino acid proline. Increasing salinity concentrations increase the content of GA3, ABA, and proline (Figure 4A,B,D). Greater activity of POD was documented at 4000 and 8000 ppm compared with non-saline water (Figure 4C). The self-grafted Strain B hybrid recorded the lowest GA3, ABA, and proline content, as well as the lowest POD activity. The higher GA3, ABA, and proline contents were registered for Edkawy combined with nano-silicon under 8000 ppm. Moreover, Edkawy combined with nano-silicon registered the greatest POD activity at 8000 ppm (Figure 4C), while the self-grafted Strain B hybrid exhibited the lowest contents of GA3, ABA, and proline with 0 ppm. Correspondingly, the self-grafted Strain B hybrid had the lowest POD activity with non-saline water. The concentration of proline was significantly increased with increasing salinity levels (Figure 4D). A higher proline content was recorded for Edkawy combined with nano-silicon under 8000 ppm.

**Figure 4.** *Cont*.

**Figure 4.** Effect of water salinity levels × rootstocks and foliar spray with (n-Si) and their interactions on (**A**) GA3, (**B**) ABA, (**C**) POD, and (**D**) proline. Vertical bars represent standard errors of the means; in each bar, values followed by different letters differ significantly at *p* = 0.05 according to the LSD test.

#### *3.7. Clustering Analysis*

Cluster analysis included all growth traits (i.e., plant height, the number of leaves per plant, fresh and dry weights of shoots and roots per plant), all yield components (i.e., the number of fruits per plant, average fruit weight, and fruit yield (kg) per plant), all mineral compositions in tomato plants (total nitrogen (N), phosphorus (P), potassium (K), magnesium (Mg), calcium (Ca), sodium (Na), iron (Fe), and zinc (Zn) concentrations), physiological traits (i.e., transpiration rate, stomatal conductance, photosynthesis, and chlorophyll reading), and activity of antioxident enzymes, gibberellic acid, abscisic acid content, and leaf proline. Figure 5 illustrates a heatmap showing the relationships among the salinity levels and grafting combinations based on the tested parameters. Heatmap analysis clearly identified the overall variations among all treatments. The grafting treatment alone increased all studied traits compared with the non-salt-stressed control. However,

foliar application of nano-silicon (n-Si) on the stressed grafted tomato exerted a positive role through decreasing oxidative injury by enhancing photosynthetic performance, increasing antioxidant enzyme concentrations, and significantly improving plant growth and yield.

**Figure 5.** Heatmap of salinity stress, rootstocks, and measured parameters of tomato samples. The differences in the response variables between all studied treatments are visualized in the heatmap diagram. Columns represent the individual response variables, while rows represent the treatments. Lower numerical values are colored blue, whereas higher numerical values are colored red (see the scale at the bottom right corner of the heatmap).

#### **4. Discussion**

For the first time, this study introduced an approach to study the effect of grafting combined with foliar application of nano-silicon to improve salt tolerance in tomato. Recently, a few studies have aimed to improve salt tolerance in tomatoes utilizing grafting [50,51]. The tomato plant is a moderately salt-tolerant plant. Plant growth parameters are a perfect indicator for evaluating several abiotic stresses on plants (Table 1). The present data indicated that grafting with foliar application of nano-silicon is an effective approach for enhancing salt tolerance in tomato, as under saline conditions the plant growth parameters of most grafted combinations and nano-silicon combinations were significantly higher than their counterparts without nano-silicon application. Considerable reductions were observed in plant height, number of leaves, and shoot and root weights with higher salinity levels, confirming trends observed in other studies [50–52]. The combination of grafting and nano-silicon application could enhance the salt tolerance of tomato plants. The results also indicated that root growth was negatively affected by salt stress, which also affected

shoot and plant height and lowered water and mineral uptake [50,52]. The trends observed were to be expected, since high salinity levels create water stress via osmotic imbalance at the soil–root interface and impair a plant's ability to take up water through the roots [53], thereby negatively impacting fresh and dry weights (Table 1). This could induce plasmolysis, which, in turn, might alter the plant's cellular and macromolecular metabolism and result in a slowing down or halting of plant growth [54]. Additionally, high salt concentrations might be damaging to both shoots and roots due to ionic toxicity brought about by excess Na+ ions, as they generate ionic imbalances within plants [50,54,55]. In this research, a high amount of Na+ documented in the shoot parts of tomato plants under salinity stress could be the result of Na+ accumulation in cellular vacuoles, whose function is to regulate osmotic balance inside the cell and maintain the photosynthetic rate at nominal levels [53]. It should be stated that the rootstock genotype had a prominent role in the performance of grafted tomato crops under stress conditions [56]. It is also worth mentioning that the data presented here suggest that the scion of the hybrid Strain B exhibited higher growth when grafted onto the rootstock of cvs Edkawy and *S. pimpinellifolium* as compared to self-grafted plants. However, there is a significant contribution made by the shoot genotype to growth [57]. Since cross-grafting involves tissue wounding and reunion, similar to what is observed in wound healing or self-grafting, it may be inferred that additional stress tolerance may be imparted by specific compatibility factors. These may include specific root/shoot signals [58], including RNA transport [59]. The foliar application of nano-silicon combined with grafting had a growth-promoting effect on unstressed tomato plants. The shoot and root fresh weights of grafted plants subjected to nano-silicon treatment increased significantly compared to the untreated grafted plants; the increase in shoot fresh weight might be described by the higher number of leaves (Table 1). The exogenous nano-silicon application may enhance the growth and yield of tomato plants [30–34], as was observed in various instances. In this study, the combination of grafting with nano-silicon application was associated with significantly higher fruit number, fruit weight, and total yield per plant values under non-saline conditions, although the smallest values were obtained at high salt levels (Figure 2). Tomato fruit yield might be described by the fruit weight and the number of fruits. Reduction in fruit weight or fruit number leads to reduced tomato fruit yield [55]. Salinity negatively impacts and decreases water and nutrient availability to the plant, causing a reduced photosynthetic rate. Such water stress is understood to result in a reduction in the number of fruits, total fruit yield, and fruit fresh weight [50,52,54]. In contrast, the data demonstrate that grafting with nano-silicon treatment counteracted salinity stress and resulted in higher fruit number, fruit fresh weight, and ultimately total fruit yield in the tomato plants compared with the controls. The appreciated effect of nano-silicon might be owed to its role in increasing RNA polymerase expression and the activity of ribosomal proteins, which stimulate stress tolerance and lower the transpiration rate and oxidative stress, thereby stabilizing the photosynthetic rate, and ultimately improving fruit yield [29,31,34]. The highest mean fruit weight was recorded for Edkawy combined with nano-silicon under 0 and 4000 ppm salt concentrations (Figure 2). Furthermore, the scion of hybrid Strain B produced a higher fruit crop when grafted onto the rootstock of cvs Edkawy and *S. pimpinellifolium* as compared to ungrafted plants, confirming the results obtained by [60]. It should be mentioned that *S. pimpinellifolium* is reputed to be rich in genes involved in biotic and abiotic stress responses in comparison with other varieties of cultivated tomato [10], which could potentially explain the trends observed. Chlorophyll is the major pigment involved in photosynthesis in plants [42]. The results indicated that under salt stress, chlorophyll readings (SPAD) were decreased, while nano-silicon addition increased chlorophyll readings (SPAD) (Figure 3C). The increase in chlorophyll readings (SPAD) with the application of nano-silicon under salinity stress might be associated with improved antioxidant defense and decreased oxidative damage due to the added nano-silicon, as observed in previous studies [31,35,47,51]. Potato plants treated with nano-silicon under salinity stress showed improved growth in terms of plant height, fresh and dry weights, and leaf chlorophyll contents [31]. In this study, transpiration rate, stomatal conductance, and photosynthesis

were significantly decreased by increasing salinity levels, while this effect was significantly alleviated by grafting along with foliar application of nano-silicon alleviates (Figure 3A,B). Transpiration rate and photosynthesis are correlated with stomatal conductance [31,34,35]. According to the results obtained, stomatal conductance showed decreased values under salinity stress, resulting in decreased transpiration rates and photosynthesis, similar to what was observed by [61,62]. The application of nano-silicon in combination with grafting is known to enhance stomatal conductance, transpiration rate, and photosynthesis, in the latter case by improving the activity of enzymes involved in photosynthesis [33,35,36,61,62]. Furthermore, the benefits of silicon nano-particles for plants grown under salinity stress are linked to an increased photosynthetic rate, stomatal conductance, and water use efficiency, all of which improve crop plant tolerance to salinity [31]. Silicon increases the stomatal conductance of plants regarding improved water status in plants under water deficit due to increased water uptake by the roots [62–65], and this increases transpiration rates and photosynthesis. Edkawy with nano-silicon and *S. pimpinellifolium* with nano-silicon recorded better stomatal conductance and photosynthesis under 4000 ppm levels. According to the results (Table 2) and previous reports, measures of tomato fruit quality, such as TSS, vitamin C content, and firmness, are changed due to salinity [55,61]. TSS content in tomato fruits is an essential factor in manipulating tomato quality. The data generated in this study indicated that TSS increased with increasing salt levels. Previous research found that TSS in tomato increased as salinity increased [11,61,66]. Foliar application of nano-silicon combined with increasing salinity increased the TSS of tomato plants, which might be referred to higher accumulations of metabolites and the direct modification of starch into soluble sugar [31,33]. In this experiment we found a significant increase in TSS in tomato fruits with grafting under the 8000 ppm level. Tomato fruit firmness increased (Table 2) with increasing salinity. Similar results have been reported elsewhere [11,67,68], and it has been found that nano-silicon increases firmness and fruit quality [11,26,39]. Increasing fruit firmness could be referred to the powerful bonding of silica to the cellulose structure [39,69]. Vitamin C (ascorbic acid) is an antioxidant that protects the body from free radical damage. According to the data, vitamin C increased with increasing salinity levels and also with nano-silicon treatment. Similar data were obtained for tomatoes [26,31,39] using nano-silicon and salinity to enhance vitamin C. The results for tomato fruits from Edkawy (Tables 3 and 4) showed that increasing salinity concentration significantly reduced N, P, K, Ca, Mg, Fe, and Zn contents in tomato shoots. On the contrary, Na content was increased with rising salt levels in tomato plants. It was reported that N, P, K, Ca, Mg, Fe, and Zn contents were lowered at high salts levels [31,55]. The results showed a decrease in tomato growth parameters and yield due to less water uptake which led to decreased mineral absorption under salinity. The higher Na amount in plants under saline conditions could be referred to the accumulation of Na inside vacuoles. On the other hand, the data for nano-silicon combined with grafting showed increased absorption of minerals under saline conditions, except Na was decreased [26]. The self-grafted Strain B hybrid showed the lowest concentrations of N, P, K, Ca, Mg, Fe, and Zn under 8000 ppm levels. Phytohormones play a major part in plant development under stress and normal conditions. Hormones such as ABA and GA have been described that regulate plant growth and development [70]. In Figure 4, it can be seen from the data that GA3 concentration reduced under salt stress but increased with the application of silicon combined with grafting. Nano-silicon mitigates the harmful effects of NaCl on plant growth by improving endogenous GA3 in soybean [31,71]. In this study (Figure 4B), ABA increased with increasing salinity levels, in a similar fashion to what has been reported in previous studies [68], and was reduced when no silicon treatment was applied, as was also observed in [72,73]. Furthermore, nano-silicon reduced Na absorbance and decreased ABA concentration in the leaves of salt-stressed plants, resulting in an increase in stomatal aperture and CO2 supply from the stomatal cavity to the CO2 fixation site [31]. In this research, POD increased with rising salt stress levels (Figure 4C). On the other hand, nano-silicon application alleviated this effect of salt. Using nano-silicon enhances antioxidant activity, hence it plays a protective function

against salt stress [28,31,59]. Compatible solutes such as proline (non-enzymatic) act as ROS scavengers and increase resistance to drought, salinity, and cold stresses [45,47,69]. In this study, the concentration of proline was significantly increased with increasing salinity levels (Figure 4D). A previous study on tomatoes yielded a similar result [74]. Higher contents of GA3, ABA, and proline were registered by Edkawy combined with nano-silicon under 8000 ppm. This could be related to the beneficial effects of nano-silicon on lipid peroxidation, plasma membrane stability, and osmolyte accumulation, all of which result in increasing concentrations of scavenging reactive oxygen species, predominantly hydrogen peroxide and superoxide [31,75].

#### **5. Conclusions**

Based on our findings, yield improvement can be achieved for tomato plants with no known tolerance to salt stress by grafting their scions on rootstocks of genotypes possessing a salinity tolerance. However, outstanding crop gain would be obtained from shoot–root exchange between salt-adapted genotypes. According to the obtained results in this study, it may be concluded that grafting combined with foliar application of nano-silicon is a unique technique for improving salt tolerance and reducing salt damage in tomato plants. Plant growth, fruit yield, fruit quality, especially vitamin C content and TSS percentage, mineral contents, and GA3, ABA, and proline levels of grafted tomato combined with foliar application of nano-silicon were significantly higher than the self-grafted Strain B hybrid under saline stress conditions. Based on these findings, it is advisable to grow the cross-grafted hybrid Strain B/Edkawy or hybrid Strain B/*Solanum pimpinellifolium* combined with foliar spray application of nano-silicon when production of tomato under high salinity conditions cannot be avoided.

**Author Contributions:** Conceptualization, E.G.S. and A.W.M.M.; methodology, E.G.S.; software, E.G.S. and M.M.E.-M.; validation, E.G.S., G.A.T., A.W.M.M. and E.G.S.; formal analysis, E.G.S. and G.A.T.; investigation, M.M.E.-M.; resources E.G.S., A.W.M.M. and M.A.A.A.; data curation, E.G.S.; writing—original draft preparation, E.G.S. and M.A.A.A.; writing—review and editing, E.G.S., A.W.M.M. and M.A.M.F.; visualization, E.G.S.; supervision, E.G.S. and M.M.E.-M.; project administration, E.G.S.; funding acquisition, E.G.S.; G.A.T. and M.A.M.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Cairo University, the Faculty of Agriculture, Giza, Egypt.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors are grateful to the Department of Vegetable Crops, the Faculty of Agriculture, and Cairo University for supplying some of the equipment and facilities used to accomplish this research.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Iodine Enhances the Nutritional Value but Not the Tolerance of Lettuce to NaCl**

**Giuseppe Maglione 1, Ermenegilda Vitale 2, Giulia Costanzo 2, Franca Polimeno 1, Carmen Arena 2 and Luca Vitale 3,\***

**Abstract:** Positive stress or essential and nonessential elements can improve nutritive values (biofortification) of edible plants. In the present study, we evaluate (i) the effect of moderate salinity on lettuce biofortification, evaluated as nutritional bioactive compound accumulation, and (ii) the role of iodine in enhancing salt tolerance by increasing photorespiration and the content of antioxidants in lettuce. Physiological (gas exchange and chlorophyll fluorescence emission) and biochemical (photosynthetic pigment and bioactive compound) analyses were performed on lettuce plants grown under moderate salinity (50 mM NaCl alone or 50 mM NaCl in combination with iodine, KIO3). Our results show that NaCl + iodine treatment improves the nutritional value of lettuce in terms of bioactive compounds acting as antioxidants. More specifically, iodine enhances the accumulation of photosynthetic pigments and polyphenols, such as anthocyanins, under salt but does not improve the salt tolerance. Our findings indicate that iodine application under moderate salinity could be a valid strategy in plant biofortification by improving nutritional bioactive compound accumulation, thus exercising functional effects on human health.

**Keywords:** nonessential elements; moderate salinity; biofortification; gas exchange; *Lactuca sativa* L.

#### **1. Introduction**

Biofortification consists in developing crops with bioavailable micronutrients in edible parts [1,2], and generally it can be achieved by breeding, agronomic, and transgenic approaches [3]. The application of positive stress (eustress) also can trigger an accumulation of bioactive compounds in edible plants [4].

Salinity, in particular an elevated salt concentration, may affect plant growth in several ways [5–8], negatively affecting the crop yield. However, at low or moderate salt concentration, yields are mildly affected or not affected at all [9], and nutritive and/or bioactive compounds can be accumulated [10,11]. Santander et al. [12] found a greater phenolic concentration and antioxidant activity in lettuce plants grown under moderate salinity (50 mM NaCl) with no effect on photosynthetic activity and plant biomass; however, severe salinity (150–200 mM NaCl) reduced the antioxidant capacity and plant biomass. An improvement of functional compounds was also found in *Perilla frutescens*, a novel food, exposed from mild to moderate salinity conditions [13]. Thus, appropriate irrigation management of horticultural crops through a mild–moderate salinity could be a means to enhance the nutraceutical value of crops because a salt-induced reshuffling of plant metabolism would increase the accumulation of nutritional bioactive compounds, thus enriching the functional quality of fresh vegetables with positive outcomes on human wellbeing.

**Citation:** Maglione, G.; Vitale, E.; Costanzo, G.; Polimeno, F.; Arena, C.; Vitale, L. Iodine Enhances the Nutritional Value but Not the Tolerance of Lettuce to NaCl. *Horticulturae* **2022**, *8*, 662. https://doi.org/10.3390/ horticulturae8070662

Academic Editors: Agnieszka Hanaka, Małgorzata Majewska and Barbara Hawrylak-Nowak

Received: 22 June 2022 Accepted: 19 July 2022 Published: 20 July 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Biofortification of vegetables to increase the mineral concentration in edible organs can also be achieved by adding essential and nonessential beneficial micronutrients [3], generally via mineral fertilizers (agronomic fortification). Several studies on mineral biofortification have been carried out in the past [14], and recently the attention has been focused on the use of iodine (I). For a long time, this element has been considered a nonessential nutrient for plants; however, now it has gained a rising interest because recent research has shown evidence for a nutritional role of iodine in vegetables [15]. Plants can utilize two iodine forms: iodide (I−) and iodate (IO3). Owing to their ability to absorb and accumulate exogenous iodine into the edible organs, horticultural crops are the best candidates to test the outcomes of I biofortification [16]. To date, it is not well understood which form of iodine is more adequate in inducing valuable results on plant biomass or inducing plant biofortification; however, it is ascertained that iodine concentrations ranging from 10−6 to 10−4 M exert positive outcomes [17].

It has been demonstrated that iodine can improve salt tolerance in plants [18], depending on variables such as the sources of iodine, their concentration, and type of application. Salt tolerance is obtained by an increase in antioxidant content, and strictly depends on the species and the form of the ion that is applied. The last aspect is very interesting because negative effects have been reported in plants fertilized with I−, whereas positive results were derived from the application of IO3 − [19]. In particular, the growth with IO3 − improves nitrogen metabolism and photorespiration in lettuce plants [20].

In the present study, we evaluate, through combined physiological and biochemical analyses: (i) the effect of the moderate salinity on the biofortification of lettuce, a species largely cultivated and consumed for human diet worldwide, assessing the nutritional bioactive compounds accumulation; (ii) the role of IO3 − in improving the salt tolerance and promoting a higher antioxidant–bioactive compound accumulation. Based on the findings of Blasco et al. [20], we hypothesize that the addition of iodine to soil leads to an increase in photorespiration and antioxidant content in lettuce plants, thereby enhancing their tolerance to moderate salinity.

#### **2. Materials and Methods**

#### *2.1. Plant Material and Growth Conditions*

Seedlings of lettuce cv Bionda liscia were transplanted 20 days after sowing (DAS) in 0.5 L pots filled with peat soil (90% peat and 10% sand, organic carbon content 31% *w*/*w*, C/N 25) and grown during autumn–winter 2021–2022. After one week from transplanting, plants were subjected to three treatments: (i) fertilization with nutrient solution (Control, EC: 1.8 dS m−1, pH: 7.5), (ii) fertilization with nutrient solution added with NaCl (3gL−1, 52 mM, EC: 7.0 dS m−1, pH: 7.5), and (iii) fertilization with nutrient solution added with NaCl (3gL−1, 52 mM, EC: 7.0 dS m−1, pH: 7.5) plus KIO3 (0.09 mg L−1, 53 μg I). Plants were grown in a greenhouse equipped with a white LED illumination system (T5-60, 14-Watt LED T5 tube, Driwei) at the following growth conditions: 100 μmol photons m−2 s−1 at the top of canopy and 16 h/8 h light/dark photoperiod. Plants were fertilized weekly—for a total of 6 weeks until the harvest by supplying 50 mL nutrient solution in which NaCl or NaCl + KIO3 were dissolved. The control received only the nutrient solution (2gL−1) with the following mineral composition: total N 20%, N-NO3 3.6%, N-NH4 3.6%, N-CH4N2O 12.1%, P2O5 40%, K2O 20%, B 0.05%, Cu 0.02%, Fe 0.4%, Mn 0.2%, Mo 0.02%, and Zn 0.02%. Each treatment consisted of 5 plants in three replicates. The plants were harvested 60 DAS, when leaves were moderately expanded. The harvest time was chosen based on a previous study [21], which demonstrated that a high post harvest quality is expected when fresh-cut lettuce presents moderately expanded leaves. At the harvesting, biometrical, ecophysiological, and biochemical determinations were carried out on leaves of different plants. The fresh vegetable quality was evaluated in terms of nutritional bioactive compounds such as: chlorophylls, carotenoids, polyphenols (flavonoids and anthocyanins), and antioxidant capacity. Soluble proteins content was also determined.

Electrical conductivity (EC) and pH were measured at harvesting in a 1:5 soil:distilled water suspension by means of a conductivity meter (Portlab 203) and pH meter (XS Instruments).

#### *2.2. Leaf Gas Exchange and Chlorophyll a Fluorescence Measurement*

A Li6400 portable photosynthesis system (Licor Inc., Lincoln, NE, USA) was used to perform simultaneous gas exchange and chlorophyll *a* (Chl a) fluorescence measurement on mature leaves. Measurements were carried out at 400 μmol photons m−2 s−1, 25 ◦C ± 2, RH 45% ± 5, and CO2 of 400 ppm. To evaluate the potential of photosynthesis and the occurrence of processes other than CO2 fixation and photorespiration, gas exchange and Chl *a* fluorescence measurements were also measured at elevated CO2 concentration (800 ppm). Gas exchange parameters: net photosynthesis (AN), stomatal conductance (Gs), intercellular to ambient CO2 ratio (Ci/Ca), instantaneous water use efficiency (ratio between net photosynthesis and transpiration—AN/Tr), were calculated by software operating in the instrument following the equation of von Caemmerer and Farquhar [22]. The fluorescence parameters in the light quantum yield of noncyclic electron transport (ΦPSII) were calculated by Genty et al. [23], while the quantum yield of regulated (ΦNPQ) and non-regulated energy dissipation (ΦNO) were calculated according to Kramer et al. [24]. The total electron transport rate (Jf) was determined as reported in Kral and Edwards [25]), and the electron transport rate to CO2 (Jc) and to O2 (Jo) was calculated according to Epron et al. [26] as:

$$\mathbf{J}\_{\rm c} = 1/3 \left[ \mathbf{J}\_{\rm f} + 8(\mathbf{A}\_{\rm N} + \mathbf{R}\_{\rm d}) \right] \tag{1}$$

$$\mathbf{J}\_{\rm o} = 2/3 \left[ \mathbf{J}\_{\rm f} - 4(\mathbf{A}\_{\rm N} + \mathbf{R}\_{\rm d}) \right] \tag{2}$$

Dark respiration (Rd) and maximum photochemical efficiency (Fv/Fm) were measured on 30 min dark-adapted leaves following the measurements in the light. Leaves were darkened through an aluminum sheet placed around the leaf, allowing air to circulate.

#### *2.3. Photosynthetic Pigments*

The content of total chlorophylls and carotenoids was evaluated following the procedure reported by Lichtenthaler [27]. Pigments were extracted in 100% ice-cold acetone and quantified by measuring absorbance at the wavelengths of 470, 645, and 662 nm. The concentration was expressed in μg cm−2.

#### *2.4. Polyphenols and Soluble Proteins*

For all the following assays, samples were preventively powdered with liquid nitrogen and stored at −80 ◦C until further analysis.

To estimate the total polyphenol content, samples (0.02 g) were extracted in 2 mL of 80% aqueous methanol following the procedure reported in Vitale et al. [28]. The extracts were kept for 1 h at 4 ◦C and then centrifuged at 11,000 rpm for 5 min. An aliquot (274 μL) was mixed with 274 μL of the Folin Ciocolteau reagent and 1.452 mL of 700 mM sodium carbonate (Na2CO3). Samples were incubated for 2 h in darkness. The absorbance was read at 765 nm and the concentration was calculated and expressed as gallic acid equivalent in mg GAE g−1 fresh weight (FW) using a gallic acid standard curve.

Total flavonoid content was quantified according to Moulehi et al. [29] and Sun et al. [30]; 250 μL of a diluted methanol sample was added to 75 μL of 5% NaNO2 (sodium nitrite, 150 μL of 10% AlCl3 (aluminum chloride), and 500 μL NaOH (1 M). Then, the mixture was adjusted with distilled water to a final volume of 1.525 mL. After absorbance determination at 510 nm, the flavonoid content was determined using a catechin standard curve and expressed as mg catechin equivalent per gram of fresh weight (mg CE g−1FW).

The anthocyanin content was analyzed according to Mancinelli et al. [31] and Chung et al. [32]. Samples (0.05 g) were extracted with acidified methanol (1% HCl) for 24 h at 4 ◦C in the dark. Samples were centrifuged and the supernatants were measured spectrophotometrically at 530 and 657 nm. The extinction coefficient of 31.6 M−1 cm−1 was used to convert absorbance values into concentrations of anthocyanins, using the following equation: anthocyanin content (μmol g−1) = [(A530 − 0.33 × A657)/31.6] × [volume (mL)/weight (g)].

The antioxidant capacity was evaluated by the ferric-reducing antioxidant power (FRAP) assay according to George et al. [33] and Vitale et al. [34]. Samples (0.250 g) were extracted with 60:40 (*v*/*v*) methanol/water solution. After centrifugation, an aliquot of extract (150 μL) was mixed with the FRAP reagents (2.5 mL of 300 mM acetate buffer pH 3.6, 250 μL of 10 mM tripyridyltriazine (TPTZ) and 250 μL of 12 mM FeCl3). After the sample incubation for 1 h in the darkness, the absorbance was read at 593 nm. The total antioxidant capacity was quantified as μmol of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) equivalents per gram of fresh weight (μmol TE g−1 FW) using the Trolox a standard curve.

Total soluble protein content was determined following Bradford [35] and Im et al. [36]. Briefly, samples (0.200 g) were extracted in 0.2 M potassium phosphate buffer (pH 7.8 + 0.1 mM EDTA) and centrifuged. The supernatant was added to the dye reagent, and the absorbance was read at 595 nm. The total soluble proteins were quantified using a bovine serum albumin (BSA) calibration curve and expressed as mg BSA eq g−1 FW.

#### *2.5. Statistical Analysis*

The statistical software SigmaPlot 12.0 was used to perform graphics and statistical analysis. Data were analyzed by one-way ANOVA followed by the Holm–Sidak post hoc test. Data shown in tables and figures are means ± standard error (SE).

#### **3. Results**

#### *3.1. Plant Growth*

Mineral fertilization added with salt (NaCl) did not alter soil pH but increased the electrical conductivity, as compared to the control (Table 1).

**Table 1.** pH and electrical conductivity (EC) in soil at the harvesting of control plants, and plants fertilized with nutrient solution added with salt (NaCl) or with salt plus iodine (NaCl + KIO3). Data are means ± SE.

Different letters indicate significant differences among treatments. *p* < 0.05.

Salt did not affect the plant growth (Table 2). The shoot biomass and the number of leaves were comparable among the treatments and no visible difference in plant appearance was observed.

**Table 2.** Main biometrical characteristics at the harvesting of control plants, and plants fertilized with nutrient solution added with salt (NaCl) or with salt plus iodine (NaCl + KIO3). Data are means ± SE.

Different letters indicate statistically significant differences among treatments. *p* < 0.05.

#### *3.2. Bioactive Compounds and Soluble Proteins*

The chlorophyll and carotenoid content was significantly affected by the treatment with salt plus iodine but not by the salt alone (Table 3). The application of only NaCl caused a 74% reduction in total polyphenols, but increased the anthocyanin content and the FRAP antioxidant capacity by 95% and 21%, compared to control, respectively. (Table 3). The NaCl + KIO3 treatment improved the total polyphenol content in lettuce leaves compared to NaCl treatment, and produced a higher anthocyanin accumulation in leaves. Conversely, the combination NaCl + KIO3 induced a reduction in antioxidant capacity by 42% compared to control and by 52% compared to NaCl treatments. No effect on soluble proteins and flavonoids was observed among the treatments.

**Table 3.** Chemical composition of control plants and plants fertilized with nutrient solution with salt (NaCl) added or with salt plus iodine (NaCl + KIO3). Data are means ± SE.

Different letters indicate significant difference among treatments. *p* < 0.05.

#### *3.3. Leaf Gas Exchange and Chl a Fluorescence Measurement*

Salt treatments did not affect leaf gas exchange both at ambient and at elevated CO2 concentration (Figure 1). According to the limitation imposed on photosynthesis by the current CO2 concentration in atmosphere, the net photosynthesis (AN) measured under elevated CO2 was significantly greater than under ambient CO2 (Figure 1a), promoting a greater instantaneous water use efficiency (Figure 1d). Under ambient CO2 concentration, salt treatments determined a significant increase in ΦPSII as compared to control (Figure 2a). The stimulation of ΦPSII by salt treatments induced a lower thermal dissipation (ΦNPQ) of absorbed light (Figure 2b) but no change in nonregulated energy dissipation (ΦNO) (Figure 2c). Under elevated CO2 the photochemical efficiency (ΦPSII) reflected the behavior of photosynthesis, resulting higher under elevated than under ambient CO2 concentration and was not affected by salt treatments (Figure 2a), as observed for ΦNPQ and ΦNO. Under ambient CO2, a higher Jf/AG ratio was measured in response to salt treatments compared to the control (Figure 2d). This result indicates an increase in electron flow toward O2 reduction (photorespiration), in contrast to CO2 reduction (photosynthesis) (Figure 2e,f), which significantly declined under both NaCl and NaCl + KIO3 treatments.

**Figure 1.** *Cont*.

**Figure 1.** (**a**) Net photosynthesis (AN); (**b**) stomatal conductance (Gs); (**c**) intercellular to ambient CO2 concentration ratio (Ci/Ca); (**d**) instantaneous water use efficiency (WUE) measured in plants fertilized with nutrient solution (C), in plants fertilized with nutrient solution *plus* salt (NaCl), and in plants fertilized with nutrient solution *plus* salt and iodine (NaCl + KIO3). Filled bars: measurements to ambient CO2; cross-linked bars: measurements to elevated CO2. Data are means ± SE. Different letters indicate statistical difference among treatments (*p* < 0.05).

**Figure 2.** *Cont*.

**Figure 2.** (**a**) Quantum yield of noncyclic electron transport (ΦPSII); (**b**) quantum yield of regulated energy dissipation (ΦNPQ); (**c**) quantum yield of nonregulated energy dissipation ((ΦNO); (**d**) electron transport rate to gross photosynthesis ratio (Jf/AG); (**e**) electron transport rate to CO2 and total electron transport rate ratio (Jc/Jf); (**f**) electron transport rate to O2 and total electron transport rate ratio (Jo/Jf) measured in plants fertilized with nutrient solution (C), in plants fertilized with nutrient solution *plus* salt (NaCl), and in plants fertilized with nutrient solution *plus* salt and iodine (NaCl + KIO3). Filled bars: measurements to ambient CO2; cross-linked bars: measurements to elevated CO2. Data are means ± SE. Different letters indicate statistical difference among treatments (*p* < 0.05).

#### **4. Discussion**

#### *4.1. Nutritional Value of Crops*

In agricultural practices, many approaches have been utilized aimed to enhance plant growth and nutritional value of crops, such as mild abiotic stress and the use of essential and nonessential elements [4,37,38].

In the present research, it was found that moderate salt levels do not modify the growth of lettuce plants, but improve their nutraceutical value, depending on the imposed salt treatment. Our findings confirm previous studies which demonstrated that eustress, such as the imposition of a mild to moderate salinity, may enhance the organoleptic components of quality in vegetables [12,39]. In our study, the vegetable quality was evaluated in terms of bioactive substances acting as antioxidants. Iodine increased the nonenzymatic antioxidants under moderate salinity, leading to an accumulation of phenolic compounds and, in particular, anthocyanins, more than under saline treatment alone. It is hypothesized that the combination NaCl + KIO3 may induce an enhancement of enzymes involved in polyphenol synthesis [14]. Similar results have been previously found in lettuce plants [32] biofortified with KIO3 at <80 μM, a concentration comparable to that used in our study. These bioactive compounds would act as antioxidants in preventing oxidative damages, and it is likely to suppose that the increase in polyphenols and, in particular, anthocyanins, was associated with plant photoprotection, because a significant increase in chlorophyll and carotenoid content occurred in plants fertilized with iodine under moderate salinity. Our results are consistent with the findings of Medrano Macías et al. [19]. It has been previously demonstrated that anthocyanin synthesis is activated by salt stress [40]; however, our results indicated that it can be potentiated by iodine. Our results also indicated that lettuce plants grown under NaCl treatment supplemented with iodine invested more energy in anthocyanin synthesis compared to the control and NaCl plants. The investment of plants in anthocyanins may have reduced the energy at disposal for the synthesis of other secondary metabolites acting as antioxidants, inducing the significant decline observed for the antioxidant capacity measured in plants fertilized with IO3 −. On the contrary, the lowest energy engaged in anthocyanin synthesis in plants grown with salt without iodine likely allowed a greater synthesis of antioxidants, enhancing the antioxidant capacity.

Our data also indicated that iodine exerted some effect on lettuce nitrogen metabolism. According to Blasco et al. [41] which reports an enhanced nitrogen assimilation and protein synthesis in plants treated with IO3 −, the increase in total chlorophyll content in lettuce plants suggests that nitrogen is assimilated in organic compounds that serve as a N donor for their synthesis.

#### *4.2. Stress Tolerance*

In our experiment, salt did not influence leaf gas exchange, evidencing that a moderate salt level may trigger plant biofortification without detrimental effects on crop yield and plant shape and architecture. Similarly, no unfavorable effect on net photosynthesis in NaCl + KIO3 plants was observed, confirming that low iodine amounts (<80 μM) are not toxic for plants [42]. Once absorbed, iodine is translocated to chloroplasts via phloem, where it exerts its effects on photosynthesis [43]. Phenolic compounds can bind iodine through an electrophilic H substitution in the aromatic ring [44], likely protecting the photosynthetic apparatus from the mineral toxicity.

Photosynthesis is compromised by salt, generally provided at high concentration in salt-sensitive plants. On the contrary, salt stress-tolerant plants activated protective responses to overcome this form of abiotic stress [8]. In both salt treatments, salt induces a greater partitioning of electrons of the photosynthetic electron chain toward processes other than CO2 assimilation, as indicated by the greater Jf/AG ratio compared to the unstressed treatment. Our data showed that the Jf/AG ratio in lettuce plants ranges from 4 to 5 under elevated CO2, a typical value indicating a nonphotorespiratory condition for photosynthetic machinery and, in turn, a negligible electron flow to O2 as an alternative acceptor to CO2. Salt, alone or in combination with iodine, induces an increase in the electron transport flow toward photorespiration (Jo/Jf), which becomes the main photochemical process other than CO2 assimilation in dissipating the absorbed light under stress. The increase in photorespiration determines a reduction in regulated thermal dissipation (ΦNPQ) sustained by the xanthophyll cycle, avoiding the rise of processes linked to photooxidation and photoinhibition of photosystems (which absence is proved by steady values of nonregulated thermal dissipation—ΦNO− among treatments). Blasco et al. [20] reported an increase in photorespiration in lettuce plants treated with IO3 −. These authors found in plants treated with high IO3 − concentration, an increase in the hydroxypyruvate reductase activity, a key enzyme of the photorespiration cycle. It is likely to suppose that under treatment with moderate salt levels, as in our experimental conditions, iodine did not enhance salt tolerance through an increase in photorespiration. Nevertheless, in both salt treatments the photorespiration rise could have provided carbonated skeletons for the photosynthetic carbon reduction cycle, thus avoiding the decline of photosynthetic activity.

In conclusion, our data show that moderate salinity improves the nutritional value of lettuce, and that iodine promotes a higher accumulation in bioactive compounds such as chlorophylls and carotenoids, polyphenols, and anthocyanins under conditions of moderate salt levels. It is likely that iodine induces a greater synthesis of anthocyanins at the expense of the synthesis of other secondary metabolites that act as antioxidants. Our data indicate that iodine does not improve salt tolerance through an ameliorating of photorespiration; however, iodine application under moderate salt levels could represent a valid strategy for plant biofortification, as it improves the bioactive compound accumulation with its valuable functional effects on human health.

**Author Contributions:** Conceptualization, G.M. and L.V.; investigation, G.M., E.V., G.C. and L.V.; data curation, E.V., F.P. and L.V.; writing—original draft preparation, L.V.; writing—review and editing, C.A. and L.V.; supervision, C.A. and L.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors thank Fabiana Fusco for her valuable help in biochemical analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Anatomical and Physiological Performance of Jojoba Treated with Proline under Salinity Stress Condition**

**M. S. Aboryia 1,\*, El-Refaey F. A. El-Dengawy 1, Mostafa F. El-Banna 2, Mervat H. El-Gobba 1, Mahmoud M. Kasem 3, Ahmed A. Hegazy 3, Heba Metwally Hassan 4, Ahmed Abou El-Yazied 5, Hany G. Abd El-Gawad 5,\*, Salem Mesfir Al-Qahtani 6, Nadi Awad Al-Harbi 6, Eldessoky S. Dessoky 7, Ismail A. Ismail 7, Mohamed M. El-Mogy 8 and El-Sayed A. EL-Boraie 9**

**Citation:** Aboryia, M.S.; El-Dengawy, E.-R.F.A.; El-Banna, M.F.; El-Gobba, M.H.; Kasem, M.M.; Hegazy, A.A.; Hassan, H.M.; El-Yazied, A.A.; El-Gawad, H.G.A.; Al-Qahtani, S.M.; et al. Anatomical and Physiological Performance of Jojoba Treated with Proline under Salinity Stress Condition. *Horticulturae* **2022**, *8*, 716. https://doi.org/10.3390/ horticulturae8080716

Academic Editors: Agnieszka Hanaka, Małgorzata Majewska, Barbara Hawrylak-Nowak and Alessandra Francini

Received: 17 June 2022 Accepted: 5 August 2022 Published: 9 August 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Abstract:** A field trial study was conducted for two consecutive seasons 2020 and 2021 in approximately 8-month-old jojoba plants to evaluate the physiological responses following salt treatment and the role of proline as a foliar application to enhance jojoba tolerance to salinity stress. Jojoba plants were irrigated once a week for four months with diluted seawater in concentrations of 5000, 10,000, and 15,000 ppm and tap water (control). Anti-stress proline was applied four times throughout the experiment, the first at the beginning of the experiment and another three times at 30-day intervals, at concentrations of 0, 300, and 450 ppm. The effect of proline treatments on jojoba plant behavior includes growth vegetative characteristics, namely plant height increase percentage (PHIP), shoot number increase percentage (NSIP), stem diameter increase percentage (SDIP), number of leaves, leaf thickness, leaf area, and fresh and dry weights of leaves, and chemical characteristics, namely chlorophyll a and b, total chlorophyll, carotenoids, leaf mineral contents (N, P, K, Na, and Cl), total phenolic content (TPC), and proline concentration. Moreover, the impacts of proline on hydrogen peroxide (H2O2), superoxide anion (O2 •−), malondialdehyde (MDA), and ion leakage (IL) under salinity stress were investigated. Briefly, proline at 450 ppm enhanced all studied growth and physiological characteristics and promoted the antioxidant system of jojoba plants compared with the control and other treatments. The anatomical structure of leaves was also examined, and favorable variations in the anatomical structure were detected in the stressed and proline-treated plants. Exogenous application of proline enhanced most of this anatomical characteristic of jojoba leaf under saline stress. In conclusion, proline as a foliar application at 450 ppm under salinity stress of 10,000 ppm enhances jojoba tolerance to salinity stress by modifying the physicochemical and morphological characteristics of jojoba plants.

**Keywords:** jojoba; proline; malondialdehyde; total phenolic content; climate change; seawater; salt stress; ion leakage

#### **1. Introduction**