**3. Results**

### *3.1. Effect of DR Stress on Tomato Seedlings Growth and Development*

Drought treatment showed a significant decline in the growth of tomato seedlings compared with CK treatment (Figure 1). The tomato seedlings, when subjected to DR stress, significantly reduced in plant height (57.52%), fresh shoot weight (FSW; 61.11%), dry shoot weight (DSW; 63.58%), fresh root weight (FRW; 63.96%), and dry root weight (DRW; 64.74%), compared with CK (well-watered) plants (Figure 2) This decline in the growth of tomato seedlings was alleviated by the exogenous application of melatonin (Figures 1 and 2). After pretreatment with ME, growth limitations caused by DR stress were improved, and less reductions in plant height (26.43%), FSW (30.25%), DSW (37.06%), FRW (35.63%), and DRW (26.13%) was observed (Figure 2).

**Figure 1.** Tomato seedlings visual demonstration under ME and DR stress. Photographs of the tomato seedlings were taken.

**Figure 2.** Exogenous supplementation of melatonin promoted the growth [plant height (**A**), fresh shoot weight (**B**), dry shoot weight (**C**), fresh root weight (**D**), and dry root weight (**E**)] of tomato seedlings under drought stress conditions. Means ± standard error, *n* = 3, significant differences are exhibited by lowercase letters (*p* ≤ 0.05), according to LSD test.

#### *3.2. Effect of DR Stress on Photosynthesis and Related Parameters*

Under DR stress, Pn, Ci, Gs, and Tr were decreased by 71.58%, 43.42%, 66.89%, and 68.04%, respectively, compared with CK seedlings (Figure 3). Conversely, when seedlings were treated with ME, the reductions of these leaf gas exchange parameters were only 44.27%, 24.59%, 43.21%, and 46.93%, respectively, as compared to the CK plants.

**Figure 3.** Exogenous supplementation of melatonin promoted leaf gas exchange [Pn (**A**), Gs (**B**), Ci (**C**) and Tr (**D**)] and level of pigment [chlorophyll a (**E**), chlorophyll b (**F**), carotenoids (**G**), and SPAD index (**H**)] of tomato leaves under drought stress conditions. Means ± standard error, *n* = 3, significant differences are exhibited by lowercase letters (*p* ≤ 0.05), according to LSD test.

A similar trend of reduction in the pigment system was observed under the DR stress condition. Chl a, Chl b, and Carotenoid content in the leaves of tomatoes were reported to be sharply reduced by 59.94%, 48.63%, and 64.74%, respectively, under DR stress (Figure 3E–G). Per contra, ME-treated tomato seedlings when subjected to DR treatment, these pigments' content significantly increased—by 86.66%, 194.68%, and 110.34%, respectively—when compared with only DR-stressed seedlings (Figure 3E–G). In DR-stressed plants, the SPAD index showed a noticeable reduction. In contrast, the ME supplementation elevated SPAD index under DR-stress (Figure 3H).

#### *3.3. Changes in Root Morphology under DR Stress*

The present study showed that DR treatment remarkably diminished the root morphological parameters, including root length (68.46%), root volume (72.95%), root surface area (72.40%), root crossings (71.02%), root tips (70.47%), root forks (66.22%), average root diameter (64.61%), and projected area (62.31%) compared with CK tomato seedlings (Figure 4). Interestingly, compared with DR stressed plants, these root characteristics were improved by 70.40-, 75.30-, 82.98-, 92.89-, 65.60-, 73.70-, 86.02-, and 52.36%, respectively, in ME pretreated tomato plants subjected to DR stress (Figure 4).

**Figure 4.** Exogenous supplementation of melatonin promoted root morphology [Root length (**A**), root volume (**B**), surface area (**C**), root crossings (**D**), root tips (**E**), root forks (**F**), average diameter (**G**), and projected area (**H**)] of tomato seedlings under drought stress conditions. Means ± standard error, *n* = 3, significant difference are exhibited by lowercase letters (*p* ≤ 0.05), according to LSD test.

#### *3.4. Relative Water Content, Proline, Soluble Sugars, and Root Activity Alternation Due to DR Stress*

After two weeks of DR stress, the RWC of DR-stressed plants was noticeably reduced by 36.10%. Conversely, ME-treatment significantly improved the RWC by 34.12% compared with DR treatment (Figure 5A). As depicted in Figure 5D, the root activity was significantly reduced in DR-stressed plants by 43.22% in contrast to normal irrigated seedlings. Nonetheless, the supplementation of ME markedly enhanced the root activity by 29.33% compared with only DR-stressed seedlings (Figure 5B). proline and Soluble sugars content were remarkably increased (124.12%, 32.59%, respectively) under the DR group compared

with CK plants. Per contra, ME treatment considerably reduced the proline content by 20.09% and 11. 24% compared with DR-stressed seedlings, respectively (Figure 5C,D).

**Figure 5.** Exogenous supplementation of melatonin promoted level of relative water content (**A**), and root activity (**B**); reduced level of proline (**C**) and soluble sugar (**D**) content of tomato seedling under drought stress conditions. Means ± standard error, *n* = 3, significant difference are exhibited by lowercase letters (*p* ≤ 0.05), according to LSD test.

#### *3.5. Oxidative Damage*

After two weeks of DR treatment, the H2O2, O2 •−, MDA, and EL levels were measured in the leaves of tomato seedlings (Figure 6). For instance, in the tomato plants under the DR environment, the H2O2, O2 •−, MDA, and EL levels significantly increased by 1.25-, 1.50-, 1.26-, and 0.99-fold, respectively, compared with well-water seedlings. Importantly, ME-pretreated plants subjected to DR-stress reduced this content only by 0.22-, 0.21-, 0.23-, and 0.22-fold, respectively, compared with the DR-stressed group (Figure 6).

**Figure 6.** Exogenous supplementation of melatonin reduces oxidative damage biomarkers [H2O2 content (**A**), O2 •− anions (**B**), MDA content (**C**), and EL (**D**)] of tomato seedling under drought stress conditions. Means ± standard error, *n* = 3, significant difference is exhibited by lowercase letters (*p* ≤ 0.05), according to LSD test.

#### *3.6. Antioxidant Enzymes Activity and Gene Expression*

The antioxidant enzymes' (APX, CAT, GR, GST, POD, SOD, DHAR, and MDHAR) activities were measured in the leaves of tomato plants (Figures 7 and 8). By exposure to DR stress, the SOD, CAT, APX, GR, and POD, enzymes activity was enhanced by 47.15-, 85.39-, 14.03-, 51.54-, and 57.77%, respectively; activity of GST enzyme was decreased by 29.51% compared to CK seedlings. It is noteworthy that when ME-treated seedlings were subjected to DR-treatment, this further elevated these antioxidant enzymes by 27.24-, 17.63-, 35.24-, 38.64-, 40.01-, and 23.02% respectively, compared with the DR-stress group (Figure 7A–D and Figure 8A,B). Moreover, subject to the DR group, the DHAR and MDHAR activity noticeably increased by 42.74- and 29.18%, respectively, compared with wellwatered seedlings (Figure 8C,D). Conversely, ME pretreatment along with DR-group,

further increased the DHAR and MDHAR activities by 25.66- and 21.26%, respectively, compared with DR-stressed seedlings.

**Figure 7.** Exogenous supplementation of melatonin promoted antioxidant enzymes [SOD (**A**), CAT (**B**), APX (**C**), GR (**D**)] and their encoding genes [*SOD* (**E**), *CAT* (**F**), *APX* (**G**), *GR* (**H**)] of tomato seedlings under drought stress condition, as shown by leaf' analysis. Means ± standard error, *n* = 3, significant difference is exhibited by lowercase letters (*p* ≤ 0.05), according to LSD test.

**Figure 8.** Exogenous supplementation of melatonin promoted antioxidant enzymes [GST (**A**), POD (**B**), DHAR (**C**), MDHAR (**D**)] and their encoding genes [*GST* (**E**), *POD* (**F**), *DHAR* (**G**), *MDHAR* (**H**)] of tomato seedlings under drought stress condition, as shown by leaf' analysis. Means ± standard error, *n* = 3, significant difference is exhibited by lowercase letters (*p* ≤ 0.05), according to LSD test.

We further measured the transcriptional levels of genes related to antioxidant enzymes (APX, CAT, DHAR, GST, GR, MDHAR, POD, and SOD) (Figures 7 and 8E–H). The tomato plants exposed to DR-stress significantly amplified the relative gene expression level of these enzymes. Per contra, ME-treated plants exposed to DR-stress added more increments in the transcriptional levels of these enzyme-related genes, when compared to DR and CK seedlings (Figures 7 and 8E–H). These outcomes suggested that exogenous ME application alleviated DR-stress tolerance by enhancing the mRNA pattern of genes of these detoxifying enzymes.

We also performed a Pearson correlation analysis of eight enzymes and their related genes. Our results revealed that the expression of the antioxidant gene was positively correlated with the drought stress condition and its expression was higher when exogenous melatonin was applied to the tomato plant (Figure 9). Similarly, the activity of antioxidant enzymes of the Halliwell Asada pathway was reported to have a positive correlation with the drought condition (Figure 9). Moreover, the expression of genes *viz*., *SOD*, *CAT*, *APX* and *GR* was reported to have a positive correlation (expressed in the blue color shade in Figure 9) with their respective enzymatic activity. However, there was also a negative but not strong correlation when SOD activity was compared with CAT, APX and GR activity. In most of the cases, the expression of eight antioxidant genes was reported to have a positive correlation with the enzymatic activities.

**Figure 9.** Pearson correlation analysis of antioxidant enzymatic activity (illustrated in bold font) and genes (illustrated in italics font) related to it.

Under DR stress, the metabolites and enzymes of the Haliwell Asada pathway were also reported to be enhanced significantly which affects the enzymatic and non-enzymatic antioxidant mechanisms in the tomato plant. Compared with CK seedlings, DR-treatment markedly increased the AsA, DHA, GSH, and GSSG content in leaves by 37.37%, 40.46, 43.51%, and 45.45%, respectively (Figure 10). Importantly, ME (100 μM) application subjected to the DR-group further increased the AsA, DHA, GSH, and GSSG content in leaves by 33.21%, 30.29%, 30.32%, and 29.16%, respectively, (Figure 10). These results suggested that antioxidant enzymes helped to reduce oxidative damage and increased the DR-stress tolerance.

**Figure 10.** Exogenous supplementation of melatonin promoted non-enzymatic antioxidant [AsA (**A**), DHA (**B**), GSH (**C**), and GSSG (**D**)] system of tomato seedlings under drought stress conditions. Means ± standard error, *n* = 3, significant difference is exhibited by lowercase letters (*p* ≤ 0.05), according to LSD test.

#### **4. Discussion**

The current study aimed to elucidate the functions applications of exogenous ME in tomato seedlings under DR stress. Plants could circumvent oxidative stress damages created by DR stress by improving their antioxidant capacity. However, if their antioxidant capacity is weak, and supplementation of some exogenous compounds having high antioxidant properties could help them to increase tolerance level to particular stress condition [18]. Amid such compounds, ME is the one posing antioxidant properties, thus it can increase stress resistance. As mentioned by Arnao and Hernández-Ruiz [45], under abiotic stresses, ME might act as a signal molecule, as it upregulates anti-stress genes and endogenous ME levels under stress conditions. Moreover, recently published reports suggested that ME has shown a protective role against DR stress in *E. japonica* [22], *A. rosea* [21], and *A. deliciosa* [20].

The tomato seedlings subjected to DR treatment significantly decreased growth characetristics. Conversely, ME supplementation notably enhanced plant growth attributes (Figure 2). Recent studies suggest that the growth and biomass production were markedly inhibited by heavy metal stress such as Ni stress in tomato seedlings. In contrast, the growth traits were markedly reinforced by ME application [17,46]. Foliar application of ME remarkably accelerated the vegetative growth of tomato seedlings under DR stress [24]. Moreover, exogenous ME ameliorated growth of *Cucumis sativus* [40] and *Trigonella foenum* [7], under DR stress condition.

During photosynthesis in the plant, carbohydrates are considered as the main supply, except for another substrate. As reported by Takahashi and Murata [47], under a stress environment, the rate of carbohydrates synthesis was reduction. Among the tomato seedlings exposed to DR stress, ME-pretreated seedlings showed an improvement in leaf photosynthesis, compared with those which were not pretreated with ME (Figure 3). Under drought stress, outcomes of the experiment carried out by Sharma et al. [26] on grafted Chinese hickory plants revealed improved photosynthetic efficiency, enhanced growth and successful recovery of chlorophyll content by the application of exogenous ME. The plethora of studies described that exogenous ME treatment recover leaf photosynthesis in *A. rosea*, *A. deliciosa* and *G. max* under DR stress [9,21,25], and *S. lycopersicum* under vanadium toxicity [43,48]. SPAD index and photosynthetic pigments including Chl a, Chl b, and Caro are necessary for the photosynthetic process which is suggested to be reduced significantly under DR stress. Interestingly, ME application robustly improved the content of the pigment of tomato seedlings (Figure 3). These results are supported by the fact that DR stress led to a reduction in leaf photosynthetic pigment, with ME foliar application alleviating these changes, and thus, ME-application might be a promising tool for mitigating DR stress in *A. rosea* [21], *Dracocephalum moldavica* [10], and *C. tinctorius* [23]. Plants obtain more energy due to increased photosynthetic capacity, which enables them to cope with environmental stresses.

Roots not only provide structural support to the aerial part of plants, but also supply nutrients and water. Thus, a plant's survival depends on its appropriate growth, development, and root functions. Drought stress significantly reduced the root growth of tomato [28]. In this work, DR treatment negatively affected the root morphological traits by decreasing root surface area, volume, length, root crossings, tips, forks, diameter, and projected area. Conversely, tomato roots pretreated with ME evidently enhanced root characteristics (Figure 4) contributing to better growth of tomato plants. Similarly, Altaf et al. [33] revealed that melatonin application dominantly enhanced the root architecture system of tomato seedling under NaCl stress. Moreover, the positive relationship between the ME application and root growth has been well-known in *S. lycopersicum* [17], *Citrullus lanatus* [49], and *Stevia rebaudiana* [50] under abiotic stresses. Interestingly, when the tomato seedlings were exposed to DR-stress, root activity declined remarkably, showing strict association to the nutrients uptake and water withholding. Per contra, ME pretreated plants repaired the roots from destruction, thus maintaining proper function of roots (Figure 5). Our study is concordant with the previously published reports where it was suggested that abiotic stress-mediated damage to the root was reported to be ameliorated by the application of exogenous ME [43].

Under drought stress, amid various accentuated responses, osmotic regulation is the most important [51]. The reduced leaf water content under DR stress leads to increment of two main osmoprotectants viz. proline and soluble sugars [52] in *S. lycopersicum* [24] and *C. cathayensis* [26]. Our results also affirm this finding (Figure 5). Nevertheless, the application of ME, particularly via root irrigation, declines levels of proline and soluble sugars. Thus, as indicated by our results, a positive turgor pressure and water balance may be maintained by ME.

Zhang et al. [40] elaborated that on the cell membrane, excess ROS caused peroxidation of pigments and lipids, ultimately upsurging the cell membranes' permeability and causing functional damages. The ROS (H2O2 and O2 •−) content, MDA, and EL have been used as oxidative damage biomarkers. In this study, the oxidative damage levels, as determined by H2O2, O2 •−, EL and MDA, were increased in tomato seedlings subjected to DR stress. Conversely, exogenous ME application effectively protected plant cells from oxidative damage (Figure 6). In a previous study, Sharma et al. [26] and Sadak et al. [27] exhibited significant lowering of MDA content and decreased oxidative stress by MEpretreatment in *C. cathayensis* and *Moringa oleifera* plants, respectively, under DR stress. Similarly, Gao et al. [53] revealed that exogenous ME application strikingly declined the level of ROS and MDA content in peach fruit. The results of our study exhibited a reduction in oxidative damage by ME application under DR stress, which is also affirmed by extended literature on various plant species such as *Citrullus lanatus* [49], *Cucumis sativus* [54], and *Stevia rebaudiana* [50], under environmental stresses. Thus, it can be concluded that ME applications can lead to reduced oxidative damage and repairing of disrupted cellular membrane induced by salinity by balancing ROS.

Antioxidant enzymes play a principal role in the defense system of plants against biotic and abiotic stress conditions. Mittler [55] described that under different stresses, increased antioxidant enzymes activities lead to potential and specific ROS scavenging. Melatonin is a multi-regulatory molecule and is recognized as a universal antioxidant [56], because it strengthens plants' antioxidant defense system and enhances tolerance, mainly by detoxifying excess ROS, which is otherwise induced by environmental stresses [57]. Melatonin noticeably improved the activity of antioxidant enzymes (APX, CAT, DHAR, GR, GST, MDHAR, POD, and SOD) and their relative genes expression (Figures 7 and 8). Altaf et al. [58] revealed that in tomato seedlings, ME surprisingly enhanced the antioxidant machinery by reduction of over-accumulation of ROS, which is primarily due to enhanced resilience to nickel toxicity. Similarly, the upregulation in relative gene expression of *APX*, *CAT*, *DHAR*, *GR*, *GST*, *MDHAR*, *POD*, and *SOD* was observed in ME pretreated

tomato seedlings under nickel toxicity [43]. Moreover, the literature exhibited significant enhancement of antioxidant enzymes' activities by ME under abiotic stresses in various plant species [26,47,59]. Furthermore, the production and accumulation balance of ROS is maintained by ME, because the performance of the antioxidative system gets boosted and the activity of antioxidative enzymes gets triggered by ME application [10,60].

On the other hand, in plant tissue, AsA and GSH is a well-known antioxidant, proving ME to be a dynamic antioxidant [61]. Wang et al. [60] reported that under environmental stresses, significant changes occur on AsA and GSH content. The present work revealed that ME supplementation predominantly elevated the AsA and GSH content. Furthermore, ME application significantly enhanced the DHA and GSSG content in tomato seedlings (Figure 10). Similarly, ME application sharply enhanced the AsA and GSH contents under Ni toxicity [58], under NaCl stress [62], and under NaHCO3 stress [63] in *S. lycopersicum*. Summarizing the discussion, our results revealed that ME alleviated the negative impact of DR stress on tomato seedlings' growth by improving photosynthesis, root architecture, antioxidant defense system and by regulating the expression of antioxidants-related genes.
