Responses Of Alfalfa (Medicago Sativa L.) To Phosphate Deficiency And Functional Analysis Of Stress-related MiRNAs

The growth of plants is seriously inhibited by the lack of available P?phosphorus?in the soil.So the sufficient available P mainly depends on external P fertilizer in agricultural production.However,the non-renewable P resources to be exhausted in the near future.This situation urges us to improve efficiency of absorption and utilization of Pi?inorganic phosphate?in plants.To analyze the strategies and regulatory pathways of efficient absorption and utilization of Pi,alfalfa with strong ability to resist low Pi?Magnum Salt?will be used to analyze the eco-physiological response to low Pi stress.And RNA genomics technology,the bioinformatics,and the molecular biology will be used to further explore the mechanisms under low Pi stress from miRNA level.The present study included three assays.?1?Screening of alfalfa varieties with low Pi tolerance:subordinate function analysis was used to comprehensively evaluate the plant's ability to resist to Pi deficiency among 25 native or imported alfalfa varieties;?2?Eco-physiological response of alfalfa to low Pi:the changes of eco-physiological indexes between normal Pi?0.5 mmol/L KH₂PO₄?and low Pi?5?mol/L KH₂PO₄?conditions were measured;?3?Identification of miRNAs response to low Pi stress:differentially expressed miRNAs under low Pi conditions were analyzed by high-throughput sequencing platform?Illumina 2500?,then potential miRNA target genes and the possible involved regulatory pathways were also predicted.The main results and conclusions were as follows.1.After 30 days of low Pi treatment,the dry weight of stems and leaves,plant height,total root length,total root square area,dry root weight,root-to-shoot ratio,Pi content,phosphate utility efficiency and activity of acid phosphatase were measured.Furthermore,9 primary indexes were transformed into3 mutual independent comprehensive indexes by using principle component analysis.The result showed that the alfalfa varieties with strong ability to tolerate low Pi stress included Aohan,Phabulous,Magnum Salt,ect.2.The results of eco-physiological responses of Medicago sativa L.cv.Magnum Salt to low Pi showed that Pi deficiency inhibited above ground and below ground biomass,and the root-to-shoot ratio was significantly increased?P<0.05?.The activity of acid phosphatase in roots was significantly enhanced.Citric,malic,and succinic acid were 3.14,1.67,and 1.09 higher than that of the control plant at 20 d under normal Pi conditions.The results showed that alfalfa increased root-to-shoot ratio,acid phosphatase activity and secretion of organic acids due to the low Pi stress.3.We constructed four libraries for Medicago sativa L.cv.Magnum Salt roots?NPR?and shoots?NPS?grown under normal-Pi conditions,and alfalfa roots?LPR?and shoots?LPS?grown under low Pi conditions.3 biological replicates per library were performed.Through high-throughput sequencing,a total of 264 known miRNAs and 157 novel miRNAs were identified.The result showed 47 and 44differentially expressed miRNAs were identified in roots and stems,respectively,in which up-regulated miRNAs included conserved miR399,miR398,miR5287,etc.,and down-regulated miRNAs included miR156,miR160,miR169,miR171,miR2643,miR396,ect.4.PsRNATarget,TargetFinder and Tapirhybrid softwares were used to predict miRNA target genes.The results showed that 909 potential target genes were predicted by consensus among the three different sets,which included transcription factors involved in growth and development containing MYB,AP2,GRAS,CCAAT-binding,and MADS-binding transcription factors and growth-regulating factor,ARF involved in root development,various protective proteins containing and Pi transporters.The results of RLM-RACE showed that cleavage sites of targets of miR156,miR160a,miR160c,miR166 and miR396 were consistent with the prediction.Gene Ontology?GO?and Kyoto Encyclopedia of Genes and Genomes?KEGG?analyses showed that miRNA targets prominently enriched in signal transducer activity,basic metabolic pathways for carbohydrates,fatty acids and amino acids;cellular response to hormone stimulus and response to auxin pathways.In the present study,based on the eco-physiological responses,differentially expressed miRNAs and their target gene under low Pi conditions,we can concluded the main responsive regulations response to low Pi stress mainly included?1?the basal response of metabolic processes of amino acids,carbohydrates and fatty acid;?2?Stress response of the formation of universal stress proteins;?3?growth and development of root systems under low Pi;?4?the regulation of Pi transporters and the specific response of secretion of organic acids.