Cucumber (Cucumis sativus L.) is a worldwide cultivated crop and China accounts for about 77% of the global production (FAO, 2016). The intensive cultivation applied in the production systems in China leads to affect negatively the plant growth through a phenomenon of autotoxicity (Bennett et al., 2012).

In general, autotoxicity results from the presence of various autotoxins secreted by roots (Yu and Matsui, 1994; Zeng, 2014). Among them, Cinnamic acid (CA) is one of the major compound which can inhibit cucumber growth (Yu and Matsui, 1994). Qiao (2013) found that CA at 0.25 mM determined a growth inhibition on cucumber seeds by reducing the germination efficiency and affecting the further development of seedlings (reduction of growth rate of roots and less lateral roots). This was found to be correlated to several CA-induced negative impacts such as, decreased ion uptake and transport, disruption antioxidant system, and imbalanced hormonal regulation. In addition, it has been proved that CA can affect the chlorophyll content in leaves and decreases photosynthetic rate (Baziramakenga et al., 1995).

Beside the importance on animal life (Nawaz et al., 2016), there is a growing evidence that melatonin (MT, N-acetyl-5-methoxytryptamine) plays a role in alleviating plant abiotic stresses, such as, extreme temperature (Fan et al., 2015), excess copper (Zhang et al., 2014), salinity (Li et al., 2017), drought (Zhang et al., 2013), and alkaline stress (Liu N. et al., 2015). The beneficial effects of MT have been proved for the improvement of several stages of the plant life cycle, such as, germination (Aguilera et al., 2015), cell division (Park and Back, 2012), root development (Passaia et al., 2014; Wang Q. N. et al., 2016), leaf senescence (Lee and Back, 2017), and crop production (Wei et al., 2014). In addition, it has been proved that MT efficiently regulates antioxidant defense systems (Szafrańska et al., 2016), increases photosynthetic efficiency (Zheng et al., 2017), reduces chlorophyll degradation, and delays leaf senescence (Liang et al., 2015) under abiotic stresses.

Cucumber (C. sativus L.) is an economically important crop in China, but it is generally sensitive to CA stress (Qiao, 2013). Although numerous recent studies have demonstrated that MT plays important roles in alleviating abiotic stresses in cucumber seedlings, such as, NaCl stress (Zhang et al., 2014; Wang L. Y. et al., 2016), water stress (Zhang et al., 2013), chilling stress (Balabusta et al., 2016; Zhao et al., 2016), high temperature stress (Xu, 2010), oxidative stress (Li et al., 2016), and nitrate stress (Zhang et al., 2017), little information is available regarding the effects of MT on plant growth under CA stress. In this study, therefore, we treated cucumber seeds and seedlings with/without MT under CA- or non-stress conditions, and then assessed cucumber seedling growth, morphology, and nutrient element and plant hormone content. The aims of this study were (1) to determine if MT pretreatment can alleviate the effects of CA stress on cucumber seedlings and (2) to detect the main factor induced by MT that alleviates inhibition of cucumber seedling growth under CA stress.

Exogenous application of melatonin has become an increasingly popular means to active plants defense mechanisms under stressful environmental conditions. However, little information is available concerning the role of MT in alleviating CA stress in plants. The present study provides evidence that the exogenous application of MT is a potential solution for improving cucumber seedling growth under CA-stress by regulating the morphological growth, and the contents of nutrient elements and endogenous hormones. Although CA inhibited cucumber seedling growth by negatively influencing most plant parameters, such as, leaf area (Figure 2), root length (Figure 3), and dry weight (Figure 4), these inhibitory effects of CA were effectively alleviated by MT application.

In this study, several lines of evidence suggested that exogenous application of MT could improve cucumber seedling growth under CA stress. First, MT application improved morphological characteristics of cucumber seedlings exposed to CA stress. These improvements were mainly achieved by MT through protecting leaves from wilting (Figure 1), stimulating leaf expansion (Figure 2), and enhancing root growth (Figure 3). Similarly, a higher health index in MT-treated tomato seedlings and a larger leaf area in MT-treated maize seedlings subjected to drought stress were observed by Liu J. L. et al. (2015) and Ye et al. (2016). According to these authors, a higher health index and leaf area enabled plants to maintain high photosynthetic capacity. Generally, roots directly reflect susceptibility or tolerance to abiotic stresses in plants. A well-developed root system can withstand adverse effects of environmental stresses (Wang et al., 2006; Arnao and Hernández-Ruiz, 2015). In the present study, MT application increased the number and diameter of lateral roots under CA stress (Figure 3). This finding is consistent with previous results (Zhang et al., 2013) in which lateral root number in cucumber seedlings was increased by MT application under water stress. Since most root morphology-related parameters (Table 1) were obviously decreased by CA under non-MT conditions but were not affected under MT conditions (Figure 3A), the improvements in root growth and development might be a major factor attributing to the alleviation of CA-stress by MT. Indeed, root morphology is an important developmental and agronomic trait that strongly influences nutrient uptake, abiotic stress resistance, and overall plant growth and development (Jung and McCouch, 2013). An overall improvement due to MT application was observed not only in our cucumber seedlings subjected to CA-stress (Figures 1–3), but also in maize seedlings subjected to salt stress (Jiang et al., 2016), tomato seedlings subjected to drought stress (Liu J. L. et al., 2015), and cucumber seedlings subjected to water stress (Zhang et al., 2013).

Second, MT application enhanced nutrient uptake by plants, especially for N, Ca, and Mg (Figures 5C,D). Among them, N is an essential mineral element that is required in the greatest amount in plants (Maathuis, 2013). The foremost function of N is to provide amino groups in amino acids, the building blocks of every protein (Maathuis, 2013). In the present work, the total N content in cucumber seedlings was increased by MT application under CA stress (Figures 5C,D). Since the total N content was strongly positively correlated with most plant morphological parameters, such as, relative growth rate, unit leaf rate, leaf dry weight, and root surface area (Table 2), N was probably one of the major nutrient elements responsible for plant morphological changes induced by MT under CA treatments. In addition to N, the Ca and Mg contents in cucumber seedlings were also increased by MT application under CA stress (Figures 5C,D). Similar results were observed in MT-pretreated cucumber seedlings under nitrate stress (Zhang et al., 2017). Ca is generally thought to have a crucial function in stabilizing cell walls and membranes (Xu et al., 2014) and is recognized as a signal in abiotic stress (Kopittke, 2012), while Mg plays an important role in plant growth and development, such as, chlorophyll biosynthesis (Hansson et al., 2013). Enhanced Ca and Mg contents in MT-treated seedlings suggested that MT might have achieved its protective effects on plant growth and development (Turk and Erdal, 2015). This could be partly supported by the positive relationships between the Ca and Mg contents and LR density/MR (Table 2), a major root morphological parameter improved by MT under CA-stress (Figures 5A,B). Moreover, MT application significantly increased the C content in the whole seedling under CA stress (Figures 5C,D), suggesting that MT might enhance aboveground C assimilation under CA-stress. Indeed, MT significantly increased the dry matter accumulation of seedlings under CA stress (Figure 4).

Third, MT improved seedling growth under CA stress by regulating endogenous hormone levels. Obviously, MT decreased the contents of ABA, MeJA, and GA₃ in seedlings under CA-stress (Figures 5E,F). More importantly, these endogenous hormones were strongly negatively correlated with most plant morphological parameters, such as, RGR, ULR, root volume, and LR density/MR (Table 2). Among these endogenous hormones, ABA is often utilized as a stress signal, which often accumulates under stressful conditions (Fujita et al., 2011). Since MT reduced the ABA content in the 2nd true leaf under CA stress (Supplementary Figure S2), MT might efficiently alleviate CA stress in seedlings. In addition to the absolute amount, the balance between endogenous hormones might play an important role in the process by which MT alleviated CA stress (Peleg and Blumwald, 2011; Ha et al., 2012). We noted that the IAA/ABA and GA₃/ABA ratios, which were strongly positively related to most plant morphological parameters (e.g., RGR, leaf dry weight, root surface area, and root volume; Table 2), were obviously increased by MT under CA stress (Figures 5E,F). Similar results were obtained in plants under other abiotic stresses such as, salinity and cold stress (Huang et al., 2015; Pompeiano et al., 2016).

Although the exogenous application of MT had a potential to improve cucumber seedling growth under CA-stress, the effects of MT on seedling growth were strongly concentration-dependent. Chen et al. (2009) demonstrated that MT stimulated Brassica juncea growth at 0.1 μM but inhibited growth at 100 μM. In this study, under non-CA stress (0 mM CA), cucumber seedling growth were suppressed by 10 μM MT (Figure 1). However, when seedling pretreated with 10 μM MT were subsequently subjected to CA stress (0.4 mM CA), the negative effects of CA on seedling growth were efficiently alleviated (Figure 1). This result suggests that the combination of two unfavorable conditions may exert a beneficial effect on plant growth.

In summary, cucumber seedling growth was generally inhibited under CA stress. Melatonin rescued cucumber seedling growth under CA stress by improving morphological characteristics, enhancing nutrient uptake, and regulating endogenous hormone levels. However, the findings presented here represent only the beginning of research on the use of exogenous MT to restore cucumber seedling growth under CA stress, and further research on the underlying physiological and molecular mechanisms is needed.

Conceived and designed the experiments: JL, YT, MQ, and LG. Performed the experiments: JL and YL. Analyzed the data: JL and YT. Wrote the paper: JL, YL, YT, and WZ.