Drought is one of the most important environmental stresses that affect plant growth and development worldwide (Chaves et al., 2003). Drought stress inhibits plant growth via many physiological processes, including chlorophyll biosynthesis, photosynthesis, energy consumption, and reactive oxygen species (ROS) metabolism (Barchet et al., 2014; Liang et al., 2019; Qiang et al., 2019). Photosynthesis is one of the physiological processes which is sensitive to environmental stresses (Johnson et al., 2015). It has been reported that drought inhibits photosynthesis through stomatal limitation and non-stomatal limitation (Barchet et al., 2014; Bai et al., 2019). Stomatal closure is one of the earliest responses of plants to drought stress, reducing transpiration and photosynthetic rate (Chaves et al., 2003). Notably, drought stress induced over-reduction of the electron transport chain, which in turn leads to photooxidation and excessive accumulation of ROS, such as superoxide radicals (O₂^•–) and hydrogen peroxide (H₂O₂) (Bano et al., 2012; Liu et al., 2012). These ROS are very reactive and damage the photosynthetic apparatus, reduce the photosynthetic efficiency, and ultimately result in cell death (Yin et al., 2005; Gupta et al., 2017; Gururani et al., 2015).

To overcome drought, plants have developed a variety of adaptation mechanisms, such as movement of leaves or chloroplasts, photorespiration, and ROS scavenging system (Yin et al., 2005; Liu et al., 2012; Basu et al., 2016). Drought resistance and adaptation in plants developed via morphological structure and physiological changes in leaves. Plants can induce stomatal closure, accumulation of compatible solutes, and biosynthesis of wax to avoid drought (Galmés et al., 2007). Additionally, the ROS scavenging system is also important for the physiological mechanisms which are supportive of drought resistance in plants. Enhanced ROS scavenging system alleviates oxidative damage and induces some molecular chaperones to protect the plant from damage (Bai et al., 2009; Basu et al., 2016). Drought stress was reported that stimulate the ROS scavenging system in apple leaves under mild to moderate stress, but that was lower under severe stress because of antioxidant enzyme damage (Liu et al., 2012). Furthermore, plants are regulated by the absorption of immobile nutrients via root systems under drought stress (Hanslin et al., 2019).

The application of biostimulators is one of the promising and practical strategies to enhance stress tolerance (Wei et al., 2015; Debnath et al., 2019). Melatonin is known as a biopromoter and plays important roles in regulating plant growth, development, and stress tolerance (Wang et al., 2012; Arnao and Hernández-Ruiz, 2014; Fan et al., 2018). In recent years, an increasing number of studies have reported the physiological functions of melatonin in plants to serve as the first line of defense against environmental stresses. Previous studies suggested that drought tolerance has increased by applying melatonin under environmental stress, such as drought, salinity, ultraviolet radiation, chilling, and heat (Posmyk et al., 2009; Galano et al., 2011; Li et al., 2012; Zhang et al., 2014; Wei et al., 2015). Stress alleviation effect of melatonin relies on various factors, including inhibiting chlorophyll degradation, improving the photosynthesis, enhancing the antioxidant system, and directly scavenging free radicals (Wang et al., 2012; Liu et al., 2015; Ke et al., 2018; Debnath et al., 2019; Asif et al., 2020).

Apple (Malus × domestica Borkh.) is one of the most economically important fruits worldwide, with the largest area of cultivation in China (Liu et al., 2012). Most apple trees are grown in arid or semi-arid regions, and drought is considered as the main environmental factor restricting the development and production of apples (Bai et al., 2019). The development of drought resistance is a priority goal in apples. Rootstocks play a vital role in productive apple orchards because of their contributions to water and nutrient uptake and abiotic stress resistance (Valverdi et al., 2019). M. hupehensis, which originates in China, is an excellent rootstock because of its resistance to abiotic stress. In addition, M. hupehensis is apomictic, resulting in uniform phenotypes and genotypes. So, it is widely used as rootstock for apple production (Huang et al., 2018). Evidence suggested melatonin is known as a biopromoter and plays important roles in regulating plant drought tolerance (Fleta-Soriano et al., 2017; Liang et al., 2019). Therefore, the objective of this work was to examine the influence of melatonin on the seedling growth, root architecture, photosynthesis, chlorophyll fluorescence of M. hupehensis exposed to drought.

Apple is known to be a large water consumer and drought stress is the major limitation for high quality and yield of apple (Atkinson et al., 1999; Liu et al., 2012). Identifying ways to produce more apples with less water consumption is essential for the high quality and yield of apples. Melatonin has been reported to play key roles in regulating environmental stresses tolerance (Galano et al., 2011; Zhang et al., 2014; Arnao and Hernández-Ruiz, 2015). In the present study, the protective effects of melatonin in drought response were investigated in M. hupehensis. The results showed that plants were wilting and that the leaves were seriously dehydrated, and almost all the measured root architecture parameters were significantly reduced under drought conditions. However, melatonin-treated plants under drought stress exhibited an effectively improved state. This agrees with the findings that melatonin-treated maize had a higher dry mass compared with the untreated plants under drought (Ye et al., 2016; Fleta-Soriano et al., 2017).

It has been reported that growth inhibition was related to photosynthesis (Galmés et al., 2007; Gururani et al., 2015; Fleta-Soriano et al., 2017). In the present study, drought stress caused a decline in all gas exchange parameters, especially for Pn on the last day of the treatment. Drought can result in photooxidation and reductions in photosynthetic efficiency (Skirycz and Inzé, 2010; Ma et al., 2015). The current results proved that exogenously applied melatonin could effectively ameliorate photosynthetic inhibition caused by drought. Additionally, exogenous application of melatonin significantly increased the proportion of medium and long root lengths. Roots play an effective role in soil water use by conveying it to the aerial parts of plants more effectively during drought (Palta and Turner, 2019). Previous research has also provided evidence for the potential role of melatonin in the rooting of plants (Li et al., 2012).

Stress-induced photosynthetic apparatus damage in the plant causes a decrease in photosynthesis, which is one of the main reasons for excessive accumulation of ROS (Bai et al., 2009; Liu et al., 2015; Gupta et al., 2017). Therefore, keeping the photosynthetic organ in good condition is important for the enhancement of plant tolerance to stress. Our study showed that drought significantly induced accumulation of H₂O₂ and O₂^•–, which resulted in higher EL. Similar results were obtained in maize (Liu et al., 2012). Also, Goharrizi et al. (2020a; 2020b; 2020c) showed that drought and osmotic stresses can increase the oxidative stress parameters, thereby elevating the content of EL. Oxidative stress caused by drought was removed via adding melatonin, which effectively protected apple plants from damage. To some extent, this could be attributed to the function of melatonin as a direct free radical scavenger (Galano et al., 2011; Arnao and Hernández-Ruiz, 2015). Substantial evidence supports that melatonin could play a vital role as an antioxidant molecule, especially under abiotic stress conditions (Li et al., 2012; Liu et al., 2015; Ye et al., 2016; Fleta-Soriano et al., 2017).

Chlorophyll plays an extremely critical role in plants, which absorb light energy and transport electrons to the reaction center during photosynthesis (Gururani et al., 2015). In plants under various environmental stresses, chlorophyll synthesis can be disrupted (Ahammed et al., 2018). Previous studies showed that exogenous melatonin helped in maintaining normal levels of chlorophyll in salt-stressed apples (Li et al., 2012). In our study, exogenous melatonin enhanced the level of chlorophyll under drought stress, suggesting that melatonin was linked to enhance the synthesis of chlorophyll. In addition, melatonin significantly decreased Chl a/Chl b at 4 d. This indicated that melatonin was able to inactive the inhibitory effect of drought stress on Chl b. The function of the photosynthetic apparatus was studied using chlorophyll fluorescence (Tang et al., 2016; Oikonomou et al., 2019). The current results showed that F0 increased under drought stress, implying that the PSII reaction center was damaged, which led to the decline of Fv/Fm. However, melatonin had a favorable effect and relieved drought-induced changes to F0 and Fv/Fm. These results suggested that 0.2 mM melatonin is an effective method to prevent damage to the PSII and to enhance the drought tolerance of apple rootstock.

Application of melatonin mitigated the growth inhibition, delayed membrane lipid peroxidation, and reduced oxidative damage in M. hupehensis under drought stress. Melatonin alleviated the adverse effects of drought stress on the growth of apple seedlings by enhancing the capacity of photosynthesis, improving chlorophyll fluorescence, and inhibiting the degradation of chlorophyll, as well as directly scavenging H₂O₂ and O₂^•– to reduce oxidative damage induced by drought stress. Thus, melatonin has been proved to improve drought stress tolerance in apple plants. Based upon the results of the current study, we present a model depicting melatonin-mediated drought tolerance of apple rootstock (Fig. 8).