Effects Of 5-azaC On Physiological Characteristics And DNA Methylation Of Wheat Seedlings Under Salt Stress

Objective:Wheat is one of the important food crops.With the continuous influence of abiotic stress on the planting environment,soil salinization has become an important factor restricting the development of wheat yield and quality.DNA methylation as a major epigenetic modification,plays a regulatory role in plant resistance to abiotic stress.Analysis of physiological characteristics and expression patterns of DNA methylation in spring wheat varieties with different salt tolerance.The relationship between the change of DNA methylation and the ability of wheat to resist adversity under salt stress was revealed from physiological and epigenetic perspectives.It provides more theoretical basis for the study of DNA methylation regulation of wheat salt tolerance.Methods:In this study,Xinchuan 11?a salt-tolerant cultivar?and Xinchun 6?a salt-sensitive cultivar?were used as materials.Wheat seeds were treated with 5-azaC?DNA methylation inhibitor?and then treated with NaCl for salt stress.Analyze some agronomic traits and physiological characteristics.The methylation of genomic DNA and specific gene sites were analyzed by methylation sensitive amplification polymorphism?MSAP?and methyltarget target region methylation sequencing,respectively.Real time fluorescence quantitative detection of relative expression level of salt tolerance gene.Results:1.5-azaC can significantly inhibited root length and reduced fresh weight and dry weight.5-azaC significantly reduced wheat plant height after salt stress,but it had no significant effect on spike length,grains per spike and grain weight per spike.Compared with salt stress alone,after treated with 5-azaC,the net photosynthetic rate?Pn?of Xinchun 11decreased significantly;transpiration rate?Tr?and stomatal conductance?Gs?decreased slowly,and intercellular CO₂ concentration?Ci?had no significant difference.Meanwhile,Tr,Gs,and Ci of Xinchun 6 decreased significantly,but there was no significant difference in Pn.After salt stress,5-azaC could significantly reduce malondialdehyde?MDA?content in wheat leaves and increased the content of proline and soluble sugar of Xinchun 6 wheat seedlings,but had little effect on Xinchun 11.Under salt stress,5-azaC significantly enhanced the activity of Xinchun 11 peroxidase?POD?,Xinchun 6 superoxide dismutase?SOD?and catalase?CAT?.2.MSAP analysis showed that the methylation levels of 5-azaC pretreated materials after salt stress showed a downward trend.After salt stress,DNA demethylation and DNA methylation occurred simultaneously in the genome.The rate of DNA demethylation increased in salt-sensitive cultivar Xinchun 6,while the DNA methylation rate decreased.The rate of DNA demethylation and DNA methylation of salt-tolerant cultivar Xinchun 11 both increased,but the rate of demethylation was higher than that of DNA methylation.5-azaC increased the rate of DNA demethylation under salt stress.Sequence analysis of DNA methylation differential modification sites found that DNA methylation modifications were present in ribosomal subunit proteins,protein kinases,and transposon sequences.3.Sequencing of three salt-tolerance related genes after treatment with bisulfite.Methylation mainly occurred at CG site,accounting for more than 40%of all types,followed by CHG,CHH types.After salt stress,Xinchun 6 first increased and then decreased,Xinchun 11 first decreased and then increased.Finally,the methylation rate of the two varieties was close to the control level.Compared with the single salt stress,Xinchun 6 had more different methylation sites than Xinchun 11.Among the 12 sites with methylation degree greater than50%,10 sites of Xinchun 6 were greater than Xinchun 11.After salt stress,the expression of TaHAK1 and TaSTK was significantly higher than that of control,and the relative expression of Xinchun 11 was always higher than that of Xinchun 6.Conclusion:5-azaC could inhibit the growth of wheat root length,caused wheat plant dwarfing,improved the antioxidant capacity of Wheat Leaves under salt stress,and alleviated the negative effects of salt stress.The sensitivity of wheat varieties to 5-azaC was related to their genotype.The demethylation of genomic DNA was mainly induced by salt stress.5-azaC increased the ratio of DNA demethylation under salt stress.Sequence analysis of methylation differential modification sites revealed that there are many metabolic pathways involved in the regulation of salt stress.Analysis of salt-tolerance related genes shows that after salt stress,the methylation rate of 5-azaC pretreatment material was similar to the control level,which indicated that 5-azaC could reduce the negative effects of salt stress on wheat and the degree of methylation play a role in gene transcription regulation.