The tea (Camellia sinensis
(L.) O. Kuntze) plant is an important economic crop that has been planted in China for nearly 2000 years [1
]. As a perennial evergreen woody crop, tea plants are often subjected to various abiotic stresses such as drought, high salt and low temperature during the growth process [2
], which are the main environmental factors that affect the geographical distribution of tea plant and limit the yield of tea [3
]. Thus, effective ways to improve the abiotic stress tolerance on tea plants are urgently needed.
Plants grown under environmental stress show the inhibition of growth and serious morphological, metabolic, and physiological anomalies ranging from chlorosis or leaf rolls to lipid peroxidation and protein degradation [4
]. Malondialdehyde (MDA) content is generally considered as an indicator of membrane structural integrity. The level of MDA content has increased significantly under abiotic stress, resulting in decreased membrane fluidity and destroyed ion homeostasis on plants [7
]. Photosynthesis, the most important physicochemical process in higher plants, is very sensitive to abiotic stress [8
]. Environmental stress can significantly affect the content of chlorophyll and the activity of key enzymes in photosynthesis [10
]. Furthermore, abiotic stress directly affects the photosynthetic system, mainly by inducing photoinhibition at both photosystem I (PSI) and PSII [11
], stress-induced inhibition of the photosynthetic electron transport results in excessive accumulation of toxic reactive oxygen species (ROS) such as hydrogen peroxide (H2
) and superoxide anion (O2−
]. The excessive accumulation of ROS promotes degradation of chlorophyll and reduces the photochemical efficiency of photosystem II (PSII) forming a vicious cycle [13
]. Moreover, the excessive ROS produced under abiotic stress is a harmful factor, which causes lipid peroxidation, enzyme inactivation and DNA damage [15
]. In response to over-induction of ROS under abiotic stress, plants employ efficient detoxifying networks, including increased activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX), and regulated contents of non-enzymatic antioxidants, including glutathione (GSH) and ascorbic acid (ASA) [5
]. Therefore, it is essential for plants to maintain the content of ROS at an appropriate level to resist abiotic stress.
-acetyl-5-methoxytryptamine) is an indole hormone involved in multiple biological processes [17
]. Since the discovery of melatonin in vascular plants in 1995 [18
], the role of melatonin related to plant physiology has attracted widespread attention. Subsequent studies have proven melatonin plays an important role in the regulation of plant growth and development [20
] and defense against abiotic stresses such as extreme temperature, excess copper, salinity, and drought [21
]. According to new findings, melatonin plays several important functions in plants. It may act as a plant growth regulator in rooting, seed germination, and delay in leaf senescence and other morphogenetic features [24
]. Furthermore, melatonin and auxin (IAA) share tryptophan as a precursor for biosynthesis and have similar structural components [26
]. However, there is no conclusive data on the metabolic transformation between melatonin and IAA in plants.
Many researchers have demonstrated that melatonin can protect plants against ROS and consequent alleviation of oxidative stress [22
]. Melatonin itself is an effective antioxidant that directly scavenges ROS. Notably, its metabolite, N
2-formyl-5-methoxykynuramine (AMFK), which has stronger antioxidant activity than melatonin, can also directly and efficiently scavenge ROS [20
]. What is more, melatonin also possesses antioxidant activity operating by modulating antioxidant enzymes and enhancing cellular non-enzymatic antioxidants [30
]. ROS-scavenging enzymes (SOD, POD, CAT and APX) and antioxidants (GSH and ASA) are necessary to provide cells with highly efficient machinery for detoxifying ROS [32
]. Recently, solid evidence was observed that plants accumulate high levels of melatonin when subjected to extreme environmental conditions [25
] and exogenous application of melatonin helps improve tolerance to stresses [34
]. As a free radical scavenger, melatonin protects the plants from oxidative stress under different environmental stresses in all species [36
]. However, it is still unclear whether such function of melatonin against abiotic stress is universal for other plant species.
Although remarkable progress has been made in investigating the role of melatonin in responses to abiotic stress, very less information about the effects of melatonin on abiotic stress tolerance in tea plants is available. In the present study, we explored the regulatory mechanisms controlling melatonin-mediated abiotic stress tolerance in tea plants and tried to understand the impact of melatonin on abiotic stress in tea plants. Hence, we analyzed the effects of melatonin on lipid peroxidation, ROS accumulation, antioxidant defense system, and photosynthetic capacity in tea seedling exposed to cold, high salt and drought stresses.
Abiotic stresses markedly inhibit plant growth via different mechanisms and result in a decrease of crop yield. In recent years, exogenous substances have been widely used to improve plant stress resistance and crop yield, among these, exogenous melatonin has emerged as a research focus in plant science [38
]. Previous studies have shown that exogenous melatonin enhances abiotic stress tolerance in some plant species including Cynodon dactylon
and Glycine max
]. Nevertheless, the mechanisms involved in melatonin-mediated tolerance to abiotic stress in tea plants still remain unknown. Thus, we examined the effects of melatonin on photosynthetic, ROS accumulation and antioxidant defense systems in tea plants under cold, high salt and drought stress. The present study indicated that the application of melatonin plays a protective role in tea plants against cold, high salt and drought stress.
The effect of exogenously applied melatonin ranges from a significant amelioration to being ineffective or even toxic [22
]. Exogenous melatonin promoted rooting at low concentration but inhibited the growth of tissue culture at high concentration [39
]. There were different concentrations applied to different plant species and organs. Our previous study indicated that 100 μM melatonin was the most effective concentration for tea leaves [40
]. In the present study, we analyzed the effects of 100 μM melatonin on tea plants under abiotic stress. To examine the photosynthesis of tea leaves when exposed to cold, salt and drought stress, we monitored the time-course of changes in values of Fv
reflects the maximum photochemical efficiency of PSII and is used to reflect the degree of damage to the photosynthetic apparatus in stress processes [41
]. Although abiotic stress significantly decreased Fv
, melatonin-treated leaves maintained a relatively higher Fv
compared with the non-melatonin treated plants (Figure 1
). This is consistent with the finding reported in tomato under cold-induced stress [42
]. It is speculated that melatonin improves photosynthetic efficiency by improving the efficiency of photosystem II in plants [30
]. These results suggest that exogenous melatonin enhanced abiotic stress resistance through increasing the efficiency of photosystem in tea plants.
Abiotic stress-induced decrease of PSII activity results in accumulation of ROS such as H2
. Although low levels of ROS are indispensable in a plant, excessive accumulations have been proven to causes lipid peroxidation, enzyme inactivation and DNA damage [15
]. Previous studies have exhibited that exogenous melatonin can decrease ROS in plants exposed to cold stress [43
], salinity stress [44
], drought stress [45
], and during senescence [30
]. In our study, the H2
content was remarkably induced by the treatment of 4 °C, NaCl and drought, but the application of exogenous melatonin alleviated abiotic stress-triggered ROS accumulation (Table 1
). Since a decreased ROS level can alleviate oxidative injury of cell membranes in melatonin-treated tea plants, we speculated that the content of MDA in these plants was lower compared with non-melatonin-treated tea plants under abiotic stress. To validate this, we measured the level of MDA in tea leaves. The results show that a lower level of MDA was observed in melatonin-treated seedlings under stress conditions (Figure 2
). MDA content is generally considered as a reliable indicator of oxidative damage reflecting cell membrane stability [11
]. This is consistent with the finding reported in cucumber under salt and cold stress [46
]. Taken together, these results show that melatonin helps to reduce membrane damage caused by over-accumulation of ROS.
To cope with oxidative stress induced by adverse conditions, plants have evolved an effective antioxidant defense system, including enzymatic and non-enzymatic antioxidants. It was reported that under oxidative stress, ROS generation increases antioxidant enzymes activities in plants [43
]. Many studies have confirmed that melatonin enhances the activity of antioxidant enzymes under abiotic stress [5
]. Consistently, in this study, oxidative stress dramatically activated the activities of SOD, CAT, POD and APX (Figure 3
). Interestingly, melatonin enhanced the activities of the antioxidant enzymes (SOD, CAT, POD and APX) further at different time points after 4 °C, NaCl and drought treatment. We also analyzed the effects of exogenous melatonin on the expression of antioxidant enzyme biosynthesis genes (CsSOD
) (Figure 5
). Exogenous melatonin significantly increased the transcription levels of CsSOD
. Other studies have shown that melatonin could regulate the corresponding genes of antioxidant enzymes in various species [32
]. Consistent with these studies, these findings confirm that melatonin stimulates the activities of the main antioxidant enzymes under abiotic stress by increasing the expression level of the corresponding genes, thereby improving the tea plant stress resistance. In addition, GSH and ASA, two important non-enzymatic antioxidants in the ASA-GSH cycle, are vital antioxidants against oxidative stress by scavenging ROS in plants [46
]. In a plant cell, O2−
can be rapidly converted to H2
by SOD, while H2
can be scavenged by an ASA and/or a GSH regenerating cycle and CAT [10
]. In the present study, GSH and ASA contents were markedly induced in melatonin-treated treatments under abiotic stress (Figure 4
). Similar studies had been reported previously [5
]. Taken together, the above results suggest that exogenous application of melatonin activated ROS detoxification of antioxidants, including enzymatic antioxidant enzymes (SOD, POD, CAT and APX) and non-enzymatic antioxidants (GSH and ASA) to maintain cellular ROS (H2
) at a relatively low level.
In conclusion, this study shows the ameliorative effects of exogenous application of melatonin on abiotic stress to tea plants. The present study provides the first evidence of the protective roles of exogenous melatonin response to multiple abiotic stresses in tea plants. To be specific, melatonin improves abiotic stress tolerance in tea plants, possibly through the enhancement of photosynthesis, the elimination of ROS and the upregulation of some key components of the antioxidant system. Our work also provides a case study that melatonin may have great potential for improving tea plant yield. However, further studies need to be conducted to provide more molecular and genetic evidence to support the mechanisms of melatonin-induced abiotic stress tolerance in tea plants.