**Melatonin Improves Drought Stress Tolerance of Tomato by Modulating Plant Growth, Root Architecture, Photosynthesis, and Antioxidant Defense System**

**Muhammad Ahsan Altaf 1, Rabia Shahid 2, Ming-Xun Ren 3,4,\*, Safina Naz 5, Muhammad Mohsin Altaf 4, Latif Ullah Khan 6, Rahul Kumar Tiwari 7,8, Milan Kumar Lal 7,8, Muhammad Adnan Shahid 9, Ravinder Kumar 7,8, Muhammad Azher Nawaz 10, Mohammad Shah Jahan 11, Basit Latief Jan <sup>12</sup> and Parvaiz Ahmad <sup>13</sup>**


**Abstract:** Tomato is an important vegetable that is highly sensitive to drought (DR) stress which impairs the development of tomato seedlings. Recently, melatonin (ME) has emerged as a nontoxic, regulatory biomolecule that regulates plant growth and enhances the DR tolerance mechanism in plants. The present study was conducted to examine the defensive role of ME in photosynthesis, root architecture, and the antioxidant enzymes' activities of tomato seedlings subjected to DR stress. Our results indicated that DR stress strongly suppressed growth and biomass production, inhibited photosynthesis, negatively affected root morphology, and reduced photosynthetic pigments in tomato seedlings. Per contra, soluble sugars, proline, and ROS (reactive oxygen species) were suggested to be improved in seedlings under DR stress. Conversely, ME (100 μM) pretreatment improved the detrimental-effect of DR by restoring chlorophyll content, root architecture, gas exchange parameters and plant growth attributes compared with DR-group only. Moreover, ME supplementation also mitigated the antioxidant enzymes [APX (ascorbate peroxidase), CAT (catalase), DHAR (dehydroascorbate reductase), GST (glutathione S-transferase), GR (glutathione reductase), MDHAR (monodehydroascorbate reductase), POD (peroxidase), and SOD (superoxide dismutase)], non-enzymatic antioxidant [AsA (ascorbate), DHA (dehydroascorbic acid), GSH (glutathione), and GSSG, (oxidized glutathione)] activities, reduced oxidative damage [EL (electrolyte leakage), H2O2 (hydrogen peroxide), MDA (malondialdehyde), and O2 •− (superoxide ion)] and osmoregulation (soluble sugars and proline) of tomato seedlings, by regulating gene expression for *SOD*, *CAT*, *APX*, *GR*, *POD*, *GST*, *DHAR*, and *MDHAR*. These findings determine that ME pretreatment could efficiently improve the seedlings growth, root characteristics, leaf photosynthesis and antioxidant machinery under DR stress and thereby increasing the seedlings' adaptability to DR stress.

**Keywords:** tomato; photosynthesis; root growth; oxidative damage; melatonin; drought; gene expression

**Citation:** Altaf, M.A.; Shahid, R.; Ren, M.-X.; Naz, S.; Altaf, M.M.; Khan, L.U.; Tiwari, R.K.; Lal, M.K.; Shahid, M.A.; Kumar, R.; et al. Melatonin Improves Drought Stress Tolerance of Tomato by Modulation Plant Growth, Root Architecture, Photosynthesis, and Antioxidant Defense System. *Antioxidants* **2022**, *11*, 309. https://doi.org/10.3390/ antiox11020309

Academic Editor: Nafees A. Khan

Received: 5 January 2022 Accepted: 26 January 2022 Published: 3 February 2022

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**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/).

#### **1. Introduction**

In recent decades, water has been considered a vital environmental factor, and its deficiency is known as drought stress (DR) which leads to restricted plant growth, yield, and quality, mainly due to various morphological, physiological, anatomical, and biochemical responses. Compared with other environmental stresses, DR stress is particularly prominent in abiotic stress [1]. Water scarcity adversely influences plant growth by reducing relative water content (RWC), root length, seedling stem, leaf area, and leaf water potential [2]. Plants exhibit multiple and interconnected responses towards DR stress. DR stress can be fatal for underdeveloped root systems of seedlings in the spring season [3]. Earlier studies revealed that DR stress can cause reduced pigment content, imbalanced ion homeostasis, decreased transpiration, stomatal closure, cell enlargement reduction, reduce canopy size, and ultimately lead towards plant death [4]. The impact of DR stress varies with the intensity and the growth stage of plants [5].

The excessive accumulation of ROS in plants under water-deficient conditions can cause oxidative damage which might lead to significant damage to the cellular organelles [6]. The ROS molecules include H2O2, O2 •−, and other oxygen-containing molecules which interact with the membrane system of the and damage mainly the macromolecules present in the cell [7]. Excessive accumulation ROS cause oxidative damage to the electron transport chain, enhance lipid peroxidation in chloroplast and mitochondria, inactivates enzyme activity, proteins, and nucleic acids, and ultimately decreases photosynthesis and yield of crops [8]. The antioxidant enzymes (APX, CAT, DHAR, GR, GST, MDHAR, POD, and SOD) and non-enzymatic antioxidants [AsA, DHA, GSH, and GSSG] molecules effectively reduce the ROS accumulation, thereby balancing the ROS synthesis and signaling to provide enhance DR-stress tolerance in plants [9]. Additionally, the accumulation of osmolyte also helps effectively against DR stress to prevent oxidative meditative damage in the plant. Soluble sugars, proline, and proteins are the most common osmolytes which interact at the cellular level by decreasing membrane permeability under mild water scarcity, thus maintaining water balances of crops under DR stress [10].

Recent trends of climate change have significantly affected crop yield and among these various abiotic conditions, DR stress is one of the main culprits [11]. To circumvent this condition, various agronomic strategies are needed for improving drought tolerance in plants which include employing phytohormones or bio-stimulators in crop production. Such phytohormones pose a positive impact on growth regulation and resilience development in plants by boosting various physiological, biochemical and molecular processes [12]. Thus, for better tolerance towards DR stress, it is of great significance to explore the effect and mechanism of these phytohormones.

Melatonin is a natural, multifunctional, nontoxic, regulatory, and universal biomolecule, having low molecular weight with pleiotropic effects in the plant kingdom [13]. Hitherto, ME is evidenced to enhance the resistance to different abiotic stresses such as DR, heat, cold, salt, heavy metals, chemicals, and pathogens [14–16]. Furthermore, ME promoted root morphology seed germination, photosynthesis, seedlings growth, delay in leaf senescence, antioxidant efficiency, fruit maturation and rhizogenesis at cellular and tissue level [17–19]. Specifically, the increased ROS homeostasis and enhanced antioxidant capacity were related to higher ME content in DR-affected seedlings, such as *Actinidia deliciosa* [20], *Althaea rosea* [21] *Eriobotrya japonica* [22], and *Carthamus tinctorius* [23].

Previous studies revealed that under drought stress, ME enhanced plant growth attributes in tomato [24], improved the root architecture system in kiwifruit [25], protected mineral balance in *C. cathayensis* [26], increased photosynthetic efficiency in *E. japonica* [22], reduced lipid peroxidation and ROS accumulation in *Moringa oleifera* [27], promoted antioxidant enzymes system in fenugreek [7], increased glutathione, and ascorbic acid content in kiwifruit seedlings [20], enhanced osmoregulatory substances in tomato [28], protected grana lamella of chloroplast in wheat [29], preserved the chloroplast structure in *Brassica napus* [30], and significantly regulated the antioxidant enzymes (APX, CAT, DHAR, GST, GR, MDHAR, POD, SOD) and non-enzymatic antioxidant (AsA, GSH) genes in *Solanum lycopersicum* and *Carya cathayensis* [19,26].

In the solanaceous family, after potato, tomato is the 2nd most important cultivated and extensively used vegetable around the world. Like other crops, water scarcity prominently causes an inhibitory effect on the physiological, morphological, biochemical, and anatomical structure of tomato [31]. Tomato is highly sensitive to water scarcity, particularly at the germination and seedling growth and seed germination stage [32]. However, information about how ME regulates the physiological, morphological, biochemical, and anatomical changes in tomato under water deficit conditions remains elusive. Therefore, in this study, we aimed to explore the impacts of ME in enhancing DR tolerance and analyze the mechanism of increased DR tolerance induced by ME. Our main focus was to investigate the photosynthetic performance, membrane damage of tomato seedlings, root architecture system, the chloroplast structure, and ROS homeostasis regulated by ME under DR stress. Precisely, we also examined enzymes related to the Haliwell Asada pathway and their genes expression. The present study has attempted to contribute to the elucidation of the biological function of ME in building resilience against DR stress.
