The Molecular Mechanism Of Melatonin Mitigating Cadmium Phytotoxicity In Radish(Raphanus Sativus L.)

Cadmium(Cd)is one of the most highly toxic heavy metals(HMs),which easily absorbed by plants and accumulated in different tissues and organs.Cadmium(Cd)pollution not only affects the crop yield and quality,but also harm human health through the food chain.Radish(Raphanus sativus L.)is an important vegetable crop,and its fleshy taproot is highly susceptible to HMs such as Cd.Plants has complex molecular mechanism of Cd detoxification and resistance,including immobilization,exclusion,chelation,and redistribution of Cd in plant cells.However,when Cd concentration is too high in the environment,the detoxification and antioxidant capacity of the plant is not sufficient to counteract the damage of HM stress.The application of exogenous plant hormones can enhance the ability of plants to resist HM stress.In recent years,melatonin(N-acetyl-5-methoxytryptamine)has been extensively studied as a plant hormone.Melatonin is a natural antioxidant,which can remove a variety of reactive oxygen species and improve the activity of intracellular antioxidant enzymes efficiently.Melatonin can enhance plant tolerance to high/low temperature,high salt,drought and UV exfoliation,as well as other abiotic stress such as chemical or metal contaminants in water and soil.In this study,the effects of melatonin on the radish seedlings were studied using the exogenous spray of melatonin method.The changes of physiological indexes of radish seedlings under Cd stress were studied.The differentially expressed miRNAs and genes of radish seedlings treated with melatonin under Cd stress were analyzed by high-throughput sequencing.These results would provide a theoretical basis for revealing the molecular mechanism of melatonin-alleviated cadmium stress in radish.The main achievements obtained were as follows.1.Melatonin treatment significantly reduced the content of malondialdehyde(MDA)in radish roots under Cd stress and increased the activities of superoxide dismutase(SOD),catalase(CAT),ascorbate peroxidase(APX),peroxidase(POD)and glutathione reductase(GR).The results showed that melatonin can effectively remove the production of reactive oxygen species in roots and leaves of radish seedlings under Cd stress,reduce the degree of membrane lipid peroxidation,protect the integrity of the membrane and enhance the antioxidant capacity of plants.Melatonin treatment can significantly increase the content of soluble protein in radish leaves and roots,increase the biomass and dry matter content,and maintain radish normal growth and development under Cd stress;The chlorophyll content,net photosynthetic rate(Pn),intercellular CO2 concentration(Ci),stomatal conductance(Gs)and transpiration rate(Tr)were significantly affected by melatonin treatment under Cd stress.Melanin can promote the accumulation of photo synthetic pigments,enhance photosynthesis,and therefore increase the radish growth and improve the Cd tolerance.Finally,melatonin can reduce the Cd content in radish leaves and roots;however,when the melatonin concentration is too high,the degree of Cd reduction is limited.in root.2.In this study,three sRNA of radish fleshy root were constructed from the control(CK),Cd-treated(Cd)and Cd plus melatonin treated group(Cd+MT),which was used for identification of miRNA and its target genes responsive to melatonin under Cd stress.A total of 87 known and 34 potential novel miRNAs were identified by high throughput sequencing.Among them,11 known and 6 novel miRNAs were identified as melatonin responsive miRNAs under Cd stress.Functional annotation indicated that the melatonin-regulated miRNAs are primarily targeted by some HM-responsive transcription factors(e.g.heat stress response transcription factor HsfA),stress-defense genes(e.g.glutathione S-transferase gene GST)and signal transduction pathway-related genes(e.g.jasmonate synthase gene JAR),and ion transport-related genes(e.g.ABC transporter gene).RT-qPCR results showed that melatonin-responsive miRNA and corresponding target genes were differentially expressed under different melatonin concentrations treatments.3.In order to identify the differentially expressed genes(DEGs)responsive for melatonin under the Cd stress,three RNA libraries of radish fleshy root were constructed using the RNA-Seq technique in control(CK),Cd group(Cd)and Cd plus melatonin treatment group(Cd+MT).Gene expression analysis showed that 343 differentially expressed genes(193 up-and 150 down-regulated genes)were obtained.Functional annotation analysis showed that many differentially expressed genes were involved in the process of reactive oxygen scavenging,metal transport and detoxification,hormone regulation,signal recognition and transduction and so on.The correlation analysis between melatonin-related response genes and miRNAs showed that most of the miRNAs and their corresponding target genes(miRNA-mRNA)showed a negative regulatory relationship in the expression trend.Some differentially expressed genes such as glutathione S-transferase GSTU,laccase gene IRX12,zinc metallopeptidase gene PXM16 and chaperone protein gene TCP-1 were involved in heavy metal response process in plants.RT-qPCR technique showed that the expression pattern of differentially expressed genes were consistent with that from the RNA-Seq analysis.