Melatonin confers plant tolerance against cadmium stress via the decrease of cadmium accumulation and reestablishment of microRNA-mediated redox homeostasis
Introduction
Cadmium (Cd), one of the most dangerous heavy metal pollutants, is toxic to animals and plants. Upon Cd stress, biomass and seedling root growth are significantly inhibited [1], [2], [3]. Strong evidence further showed that Cd causes the inactivation or denaturation of proteins, thus resulting in oxidative stress at cellular levels [4], [5]. In response to Cd stress, plants have evolved complex mechanisms, including restrict Cd influx, and promoting Cd efflux and chelation, etc [6], [7]. Among these, the decrease in Cd uptake is an important step for eliminating Cd toxicity.
In plants, the ATP-binding cassette transporter (ABC transporter), a Cd extrusion pump, confers heavy metal resistance [8], [9]. Plant natural resistance-associated macrophage protein (Nramp) also participates in cellular Cd uptake and Cd transport within plants [10], [11]. In alfalfa plants, ABC and Nramp6 are homologous genes of Arabidopsis PDR8 (AtPDR8), Arabidopsis Nramp6 (AtNramp6), respectively. Genetic evidence showed that AtPDR8-over-expressing plants were more resistant to Cd than the wild-type and had lower Cd contents [9]. Meanwhile, a null allele of AtNramp6 was more tolerant to Cd toxicity, a phenotype that was reverted by expressing AtNramp6 in the mutant background [10].
On the other hand, the accumulation of reactive oxygen specie (ROS) induced by Cd stress might have a dual role in the process of plant cells, including acting as toxic by-products of metabolism, and key regulators of growth, development, and defense pathways [12], [13]. For example, ROS-induced lipid peroxidation and oxidative damage in other macromolecules, and/or cell death may occur [1], [2], [12]. To keep the redox homeostasis, plant cells possess certain enzymatic antioxidant defence systems (including superoxide dismutase, SOD; catalase, CAT; guaiacol peroxidase, POD; ascorbate peroxidase, APX; etc) responsible for ROS scavenging.
It was well-known that microRNAs (miRNAs) are a class of non-coding small RNAs of approximately 21–24 nucleotides, which could modulate gene expression at the transcriptional and post-transcriptional levels via guiding target mRNA cleavage or translational inhibition [14]. In recent years, many miRNAs have been demonstrated to have important regulatory functions in plant growth, development and stress responses [15], [16]. For example, miR398 was the first miRNA identified to be regulated by oxidative stress. Its targets are the Cu/Zn superoxide dismutases (CSD) enzymes: cytosolic CSD1 and plastidic CSD2 [17].
Melatonin (N-acetyl-5-methoxytrytamine) was firstly discovered, and isolated from the bovine pineal gland in 1958 [18]. In animals, melatonin is involved in various physiological functions, such as anti-inflammatory actions [19], innate immunity [20] and, Alzheimer’s disease [21], etc. In plants, research efforts over the past decade have focused on determining its many roles in plant physiology. Melatonin was discovered in plants in 1995 [22]. To date, it has been found in various plants, including Arabidopsis, rice, cucumber, tomato, tobacco, cabbage, apple, alfalfa, bermudagrass, mustard, etc [23], [24]. Studies have discovered that melatonin is synthesized via four continue enzymatic reaction from tryptophan, requiring tryptophan decarboxylase (TDC), tryptamine 5-hydroxylase (T5H), N-acetylserotonin methyltransferase (ASMT), and serotonin N-acetyltransferase (SNAT) [25], [26]. Various plant species rich in melatonin have shown a higher capacity for stress tolerance. For example, overexpression of MzASMT in transgenic Arabidopsis plants exhibited high levels of melatonin and conferred resistance to drought transgenic [27]. Melatonin levels in the snat knockout mutant lines were 50% less than in wild-type of Arabidopsis, which exhibited susceptibility to pathogen infection [28].
The roles of melatonin in plants are mainly included protecting plants from adverse environmental conditions, such as heavy metals, cold, high temperature, salt, aging, drought, and peroxidizing herbicides [29]. Above beneficial responses in plants were mostly attributed to the antioxidant behaviors of melatonin. However, molecular and genetic evidence supporting the detailed mechanism of melatonin in plants is limited. In this report, we generated transgenic Arabidopsis lines overexpressing alfalfa SNAT (MsSNAT) to investigate the mechanisms of melatonin involving in plant Cd tolerance. Cd-induced melatonin production was observed both in alfalfa seedling and transgenic Arabidopsis. More importantly, the pretreatment with melatonin could alleviate Cd-induced seedling growth inhibition in alfalfa. The potential roles of endogenous melatonin counteracting Cd stress, and decreasing Cd accumulation by modulating Cd transporter in plants, were discovered. Our results further showed that melatonin-conferred Cd tolerance is partially associated with the changed expression levels of miR398 and its target genes, including CSD1 and CSD2. Thus, redox homeostasis was reestablished so as to adapt to Cd stress.
Section snippets
Plant materials, growth conditions and treatments
Commercially available alfalfa (M. sativa L. cv. Biaogan) seeds were surface-sterilized with 5% NaClO for 5 min, rinsed comprehensively in distilled water and germinated for 1 day at 25 °C in the darkness. Uniform seedlings were cultured with quarter-strength Hoagland solution in the illuminating incubator (14 h light with a light intensity of 200 μmol m−2 s−1 irradiation, 25 ± 1 °C, and 10 h dark, 23 ± 1 °C). Seedlings pretreated with or without melatonin were incubated in quarter-strength Hoagland
Cd-induced inhibition of alfalfa seedling growth and endogenous melatonin production
To analysis the toxic threshold of cadmium (Cd) stress on growth performance, alfalfa seedlings were exposed to 0, 50, 100, and 200 μM CdSO4 for 3 d. As shown in Fig. 1A, exposure of seedlings to Cd resulted in marked decreases in the elongation of seedling roots and fresh weight of root tissues. For instance, when treated with 100 μM Cd, the root elongation and fresh weight were decreased by 51% and 52%, respectively. Interestingly, melatonin contents were progressively increased in root tissues
Discussion
Cadmium (Cd) is a non-essential and toxic metal for living organisms, causing visible symptoms, renal dysfunction and cancers [36]. Although numerous studies have characterized the protective roles of melatonin in the alleviation of Cd toxicity in animals [37], [38], [39], corresponding mechanism in plants has not been largely unknown. In this study, by using pharmacological, genetic and molecular approaches, we revealed that melatonin improved plant adaptation to Cd stress by decreasing Cd
Acknowledgments
This work was partly supported by the Natural Science Foundation of Jiangsu Province (BK20130683), China Postdoctoral Science Foundation (2014M551608), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the National Natural Science Foundation of China (J1210056 and J1310015).
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