Effects Of Melatonin On Antioxidant Capacity In Chlamydomonas Reinhardtii And Naked Oat Under Salt Stress

Melatonin?MT;N-acetyl-5-methoxytryptamine?has antioxidant activities in animals and protects plants from various biotic and abiotic stressors.MT is a pleiotropic signaling molecule that has been demonstrated to play an important role in plant growth,development,and regulation of environmental stress responses.The study data showed that MT induced plant antioxidant enzymes activity and enhanced plant stress resistance.And MT also could enhance plant stress resistance in green algae.However,there are still many unknown mechanisms for the antioxidant protection of MT.Known as“green yeast”Chlamydomonas reinhardtii is a unicellular green algae that has a fast growth rate and simple molecular genetic background.It is used as a model organism for the study of plant functional genes and secondary metabolites.Naked oat?Avena nuda L.?is an annual herbaceous crop in the family Poaceae.Naked oat is one of high nutritional value cereals and is a unique food crop in Chinese alpine regions.Naked oat is extremely nutritious and medically valuable,and it is also used as both a food and a grass for animal husbandry production.However,abiotic stressors directly limit growth of naked oat,which restricts effective use and development of the plant.The study of MT in unicellular algae and naked oat is still very limited under salt stress.In the present study,C.reinhardtii and naked oat were treated with NaCl stress to study the effects of MT on antioxidant capacity in plants.On the one hand,transgenic lines were obtained by glass bead transformation method,thereby analyzing the growth and antioxidant enzyme activity.On the other hand,effects of MT on antioxidant activity in naked oat seedlings under salt stress.These results provide a reference on the study of MT function in plant.The main results are as follows:?1?Construction of antisense expression vector pDBle-aanat in C.reinhardtii:The plasmid pMD18-aanat and pDBle were digested with NdeI and EcoRI,and then the targeted fragments were recovered by DNA gel recovery kit.The pDBle fragment and the targeted gene fragment were inversely connected to transform E.coli DH5?competent cells.The target strain was also amplicated by PCR,double digestion and sequenced.The result showed that the constructed expression vector pDBle-aanat was correct.?2?The transgenic algae lines were obtained by glass bead transformation methods:The transformed lines were obtained by glass bead transformation methods,screened and cultured on TAP solid medium containing bleomycin?Ble?resistance.The DNA and RNA of the transformed C.reinhardtii were extracted.Ble gene was detected by PCR,and AANAT gene expression level was performed by qRT-PCR.The positive C.reinhardtii ARn?n is a different transgenic line?was finally determined,and it was proved that the aanat gene was successfully integrated into the genome of C.reinhardtii and downregulated the expression level of AANAT gene.?3?Analysis of the growth condition of transgenic strains:The OD value of WT line and transgenic lines was determined.The data showed that the growth curve of the transgenic lines were basically consistent with the WT line,except that the cell density of the transgenic lines was lower.However,the average MT content in transgenic lines was 10.37pg/mL,which was lower than that of WT line?MT content was 11.86 pg/mL?.It demonstrated the AANAT gene silencing in transformed lines reduced MT biosynthesis.?4?Physiological response of transgenic lines?endogenous MT?in salt stress:?1?The intracellular MT concentration of different strains was determined.It was found that the concentration of MT in the transformed line AR₃ was significantly lower?p<0.05?than that of the non-transformed line;?2?The ability of AR₃ line to scavenge reactive oxygen species?ROS?and malondialdehyde?MDA?was weaker than that of WT line when they were pretreated with two concentrations of NaCl?50 mM and 100 mM?;the antioxidant enzymes activity was decreased.Similarly,the expression level of antioxidant enzyme genes was downregulated.These results demonstrated that AR₃ had a reduced ability to scavenge ROS and restore cell damage.?5?Protective effect of exogenous application of MT in naked oat under salt stress:?1?Under 150 mM NaCl stress,plant height,stem thickness,plant fresh weight,plant dry weight,chlorophyll content,leaf area and leaf volume in the plants pretreated with 50 or100?M MT followed by salt stress were increased,respectively,than those in the control plants after NaCl stressed;and the average expression level of chlorophyll biosynthesis gene?ChlG?was upregulated 6.40-fold.These results demonstrated that MT might promote plant's growth.?2?Under 150 mM NaCl stress,hydrogen peroxide?H₂O₂?,superoxide anion(O₂^·-)and MDA contents in naked oat seedlings decreased averagely after pretreatment with two concentrations of MT;superoxide dismutase?SOD?,peroxidase?POD?,catalase?CAT?,and ascorbate peroxide?APX?activities increased.These results demonstrated that MT played an important role in improving the antioxidant capacity in plant by removing intracellular ROS and increasing the antioxidant enzymes activity.?3?Under 150 mM NaCl stress,proline content increased after pretreatment with two concentrations of MT;while soluble protein content decreased;the expression level of lipid peroxidase genes?LOX and POX?was upregulated.These results demonstrated that proline might be the major osmotic regulator in naked oat,and they also showed that MT could induce the expression of genes related to salt tolerance,thereby increasing plant's tolerance to salt stress.?4?Under 150 mM NaCl stress,the expression level of mitogen-activated protein kinase?MAPK?genes?Asmap1 and Aspk11?was upregulated and the transcription factors?TFs?NAC,WRKY1,WRKY3,and MYB genes were upregulated by pretreated with two concentrations of MT.These results revealed the important role of MT in the relief of salt stress.It is speculated that MT might have a similar effect with brassinosteroids?BRs?.MT might induce MAPK cascades through H₂O₂ signaling pathway to regulate the expression of genes involved in salt stress-related signal transduction,thereby increasing the plant's tolerance.Whether it is a low expression study of the MT biosynthase gene AANAT or an exogenous MT study,it is confirmed that MT had an antioxidant function.