Responses Of MiR408 And Its Targets To Nitrogen Stress In Medicago Truncatula

Alfalfa(Medicago sativa L.)is an important leguminous forage with high forage value,wide ecological adaptability and high yield.Alfalfa nodule can fix nitrogen in a high efficiency.However,it has to face the situation of nitrogen deficiency sometimes.when nitrogen supply is insufficient,the nodule growth is inhibited and alfalfa growth and development are affected seriously.As an important functional microRNA,miR408 plays a crucial role in copper homeostasis and cell wall biosynthesis in plant cells.Moreover,it has been suggested to respond to nitrogen deficiency as well.This research employed Medicago truncatula(R108)as a model legume species to study the changes of phenotypes and gene expression of M.truncatula as well as the alteration of MtmiR408 and its target gene under the condition of nitrogen stress.Furthermore,pre-miR408 overexpression and miR408 mimicry vectors were constructed,respectively.And the transgenic research of M.truncatula was carried out.This study will provide basic information for further analysis of molecular mechanism of alfalfa in response to nitrogen deficiency stress.The main findings are listed below.1,The phenotype of M.truncatula was changed under nitrogen deficiency including the reduced biomass,increased root length,reduced chlorophyll content,and elevated anthocyanin content.Since nitrogen is an important component of plant chlorophyll,we theorized that nitrogen deficiency may result in biomass changes by affecting photosynthesis of M.truncatula.2,The expression level of miR408 was decreased in M.truncatula after 12 h of nitrogen deficiency treatment.The miR408-targeted genes,like blue copper protein,plastocyanin,and phosphate-responsive protein genes were significantly changed responsive to nitrogen deficiency treatment.Their expression patterns,however,were not exactly consistent with that of miR408 in different tissues/organs of M.truncatula.It suggests that these target genes are not only regulated by miR408,but may also be co-regulated by other genes.3,M.truncatula samples taken at 0 h,12 h,24 h,and 48 h after nitrogen deficiency treatment were subjected to RNA-seq.A large number of differentially expressed genes(DEGs)were retrieved from the transcriptome data.Moreover,the DEGs were different among the samples treated with different time and different parts of M.truncatula.For example,the number of DEGs examined at 12 h after nitrogen deficiency treatment reached 2,276.In addition,the GO and KEGG enrichment analysis showed that the DEGs were involved in multiple pathways including nitrogen metabolism,transporter protein,and photosynthesis,suggesting a synergistic responses to nitrogen deficiency in M.truncatula.4,The vectors containing pre-miR408 overexpression and miR408 minicry cassettes,respectively,were constructed,and then 33 transgenic M.truncatula plants were generated which will be used to study the detailed functions of miR408 in future.This study laid a solid foundation for further understanding the molecular regulation mechanism of nitrogen deficiency in alfalfa.