Identification Of Drought Memory-related MiRNAs And Their Biological Function Analysis In Wheat

Wheat is the essential crop globally,providing 30% of the global food source.Various abiotic stresses,including drought,have resulted in significantly reduced wheat yields.Improving the drought resistance of wheat and breeding drought-tolerant high-quality wheat is an urgent need to promote stable and increased wheat production.Due to the fixation of plants and the variability of their growing environment,plants may experience repeated environmental stresses throughout their life cycle.The molecular mechanisms of plant response to similar abiotic stress(drought exercise)are different from its first encounter.Drought memory is a conserved drought resistance mechanism developed by plants during long-term evolution.Early drought stress training makes plants develop a stress memory that allows them to respond to drought more positively and aggressively when exposed to drought stress again.As an essential transcriptional regulator,miRNAs have important regulatory roles in plant growth,development,and response to adversity.Therefore,this study combined miRNA and degradome sequencing approaches to systematically identify wheat drought memory-related miRNAs and their target genes in wheat.And the functions of crucial candidate drought memory-related miRNAs and the regulatory networks were explored.And the regulatory mechanisms of drought memory-related miRNAs were preliminarily resolved in wheat.The main findings are as follows:(1)Drought memory function was produce by drought exercis can enhances drought resistance in wheat seedling.Compared with the direct drought treated wheat,the drought memory treated wheat may need lower proline accumulation and higher photosynthetic rate to maintain cell homeostasis in the initial stage of more severe drought stress.Still,with the extension of drought time,the proline content of the direct drought group is significantly higher than that of the drought memory group,which indicates that the drought memory treated wheat only needs less proline to maintain cell homeostasis.(2)A total of 1481 miRNAs were identified by miRNA sequencing,including 644 known miRNAs and 837 conserved miRNAs,and a total of 146 novel miRNAs were predicted after eliminating low-expressed miRNAs.228 direct drought response-related miRNAs were identified by differential expression analysis,including 82 known,60 conserved and 86 novel miRNAs.There are 195 candidate drought memory-related miRNAs that were finally identified,including 186 drought memory-specific miRNAs and nine significantly differentially expressed miRNAs shared by direct drought and drought memory.(3)Among 195 candidate drought memory-related miRNAs,64 drought memoryresponsive miRNAs were identified by degradome sequencing.10 key drought memoryrelated miRNAs including tae-miR408＿L-1、tae-miR171a、tae-miR164a-5p、tae-miR396c-5p、tae-miR9778、tae-miR408＿L-1、tae-miR9676-5p、tae-miR16s2p39、tae-miR16s-miR396-MIR9-3p、PC-3p-5049＿3565 and their target genes were further verified by RT-q PCR.The results showed that drought memory-related miRNAs strongly responded to the drought by regulating the expression of their target genes.Drought memory can regulate multiple signaling pathways in response to drought stress,such as growth hormone-activated signaling pathways,calcium transport,heavy metal transport,glutathione biosynthesis,protein modification,electron transport,energy metabolism,amino acid metabolism,and plant growth and development.A drought memory-induced drought response model in plants was proposed:drought memory-related miRNAs can improve plant drought resistance by promoting polysaccharide catabolism and soluble sugar accumulation and regulating proline homeostasis.(4)The precursor sequence of tae-miR531＿L-2 was cloned from the wheat genome and overexpressed in Arabidopsis thaliana.The RT-q PCR analysis found that tae-miR531＿L-2had a strong drought,freezing,and salt stress responses.In addition,tae-miR531＿L-2 could significantly overexpress and improve the seed germination rate and seedling stage drought resistance of transgenic Arabidopsis under drought stress.